Search

Current clinical application of platelet-rich plasma is showing a trend toward multiple treatments. The goal of this study was to show the benefit of interval platelet-rich plasma application in the healing and recovery of human tenocytes using an in vitro cell model. Eight volunteers (6 men and 2 women) were included in this study (mean±SD age, 31.6±10.9 years). Venous blood was collected from new blood draws at 3 different times. Two blood products were prepared on each day of treatment: platelet-rich plasma derived from a single-spin process (PRPSS) and platelet-rich plasma derived from a double-spin process (PRPDS). The study had 2 limbs: 2-day and 4-day intervals. Cell proliferation, measured as disintegrations per minute, was then examined via a radioactive thymidine assay. In the 2-day-interval group, the difference in disintegrations per minute between days 0 and 2 in the PRPSS group reached statistical significance (P =.006). In the PRPDS group, statistical difference was seen between days 0 and 4 (P=.001) and between days 2 and 4 (P=.030). In the 4-day-interval group, the difference in disintegrations per minute between days 4 and 8 in the PRPSS group reached statistical significance, showing a decrease in cell proliferation (P =.013). In the PRPDS group, a statistical difference was seen between days 0 and 8 (P=.021), also showing a decrease in cell proliferation. The greatest effect of platelet-rich plasma, which has a positive effect on tenocyte proliferation and recovery, is seen on initial application. Its effect is diminished with repetitive application, and this finding leads to questioning of the efficacy of interval platelet-rich plasma dosing., (Copyright 2015, SLACK Incorporated.)

The integration of tendon into bone occurs at a specialized interface known as the enthesis. The fibrous tendon to bone enthesis is established through a structurally continuous gradient from uncalcified tendon to calcified bone. The enthesis exhibits gradients in tissue organization classified into four distinct zones with varying cellular compositions, mechanical properties, and functions in order to facilitate joint movement. Damage to tendinous insertions is common in the field of orthopaedic medicine and often involves surgical intervention that requires the attempted recreation of the natural organization of tendon into bone. The difficulty associated with recreating the distinct organization may account for the surgical challenges associated with reconstruction of damaged insertion sites. These procedures are often associated with high failure rates and consequently require revision procedures. Management of tendinous injuries and reconstruction of the insertion site is becoming a popular topic in the field of orthopaedic medicine.

Studies using in vitro cell models enable evaluation of the effects of different PRP products under very controlled and standardized conditions. Therefore the results of such studies build the basis for understanding the variable results of clinical studies on the use of PRPs. The main lessons learned through the use of in vitro cell models are that many different PRP products exist and researchers have to report on component variation within each product. These different products may have distinctive effects on the various cells treated in musculoskeletal injuries; therefore, some products might be more beneficial in certain indication than others. In its utilization in cell models, PRP may generate a variety of positive effects on cell proliferation, recovery, and inflammatory response. There might also be a benefit to adding PRP to current pharmacological therapies (e.g. corticosteroids) to prevent their commonly known negative effects on e.g. tendon and cartilage tissue.

Introduction: cell-based tissue engineering strategies using human mesenchymal stem cells (hMSCs) may help to augment tendon healing. To further investigate the in-vitro behavior of this cell population, we investigated low oxygen culture levels, and growth factor supplementation and their effect on expression of tendon extracellular proteins and cell proliferation., Materials and Methods: bone marrow aspirate (BMA) was harvested during arthroscopic rotator cuff repair. Characterized hMSCs derived from BMA were incorporated into 3-dimensional tissue engineered constructs (TECs). TECs were analyzed by frozen sections with immunohistochemistry for cell density, collagen I and collagen III expression., Results: growth factor stimulation and low oxygen increased cell density within TECs. Low oxygen and addition of growth factors to culture media demonstrated an increase in collagen I and III expression, both in ambient oxygen conditions and low oxygen conditions., Conclusion: low oxygen and TGFβ3 demonstrated a positive effect on cell number, and type I and III collagen expression in 3D culture environments.

Purpose: This study investigates the antibacterial properties of two different platelet-rich plasma (PRP) platelet concentration preparations (PRPLP and PRPHP) through a time-kill assay., Methods: Two different PRP preparations - a single spin process yielding lower white blood cells and platelet concentration (PRPLP) and one yielding high platelet and white blood cell concentration (PRPHP) - were obtained from 2 individuals. PRPLP, PRPHP, phosphate buffered saline (PBS), whole blood and Cefazolin were added to experimental reaction tubes, each containing a single bacterial inoculum of Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epi), methicillin-resistant Staphylococcus aureus (MRSA) or Propionibacterium acnes (P. acnes). Two dilutions (1:1,000, and 1:10,000) were plated in duplicate tubes, along with positive (blood and PBS) and negative (Cefazolin) controls and assessed at five time points (0, 1, 4, 8 and 24 hours)., Results: After centrifugation, platelet count of PRPLP was 386 ± 65.5 × 103/?L and PRPHP was 867 ± 234.4 × 103/?L. Both PRP products showed a significant decrease (p<0.05) in bacterial growth at 8 hours compared to whole blood., Conclusion: The application of PRPLP and PRPHP showed a significant decrease in bacterial growth after 8 hours for S. aureus, S. epi, MRSA and P. acnes compared to the whole blood control group. S.epi, MRSA, and P. acnes also showed a significant decrease in bacterial growth after 24 hours. Despite differences in platelet concentration and WBC concentration, no difference in antibacterial activity was seen between the two preparations.

Tendon imaging plays a critical role in evaluating tendon diseases and injuries including mechanical, degenerative, and overuse disease, inflammatory enthesitis, as well as partial and full thickness tears. Ultrasound and magnetic resonance imaging (MRI), each with unique benefits and limitations, are commonly utilized to assist in diagnosing these diseases and conditions. This review delineates important structural properties of tendon and biochemical changes occurring in tendon pathology. This review also examines commonly injured tendons including tendons of the elbow, tendons of the rotator cuff of the shoulder, hip abductor tendons, patellar tendons, and the Achilles tendon to help clinicians better recognize tendon disease. Finally, this paper introduces several emerging imaging techniques including T2 mapping, ultra-short echo time MRI, and sonoelastography as ways in which tendon imaging and evaluation may be improved.

Purpose: The purpose of this study was to examine, in vitro, the cellular response of human mesenchymal stem cells (MSCs) to sample types of commercially available scaffolds in comparison with control, native tendon tissue (fresh-frozen rotator cuff tendon allograft)., Methods: MSCs were defined by (1) colony-forming potential; (2) ability to differentiate into tendon, cartilage, bone, and fat tissue; and (3) fluorescence-activated cell sorting analysis (CD73, CD90, CD45). Samples were taken from fresh-frozen human rotator cuff tendon (allograft), human highly cross-linked collagen membrane (Arthroflex; LifeNet Health, Virginia Beach, VA), porcine non-cross-linked collagen membrane (Mucograft; Geistlich Pharma, Lucerne, Switzerland), a human platelet-rich fibrin matrix (PRF-M), and a fibrin matrix based on platelet-rich plasma (ViscoGel; Arthrex, Naples, FL). Cells were counted for adhesion (24 hours), thymidine assay for cell proliferation (96 hours), and live/dead stain for viability (168 hours). Histologic analysis was performed after 21 days, and the unloaded scaffolds were scanned with electron microscopy., Results: MSCs were successfully differentiated into all cell lines. A significantly greater number of cells adhered to both the non-cross-linked porcine collagen scaffold and PRF-M. Cell activity (proliferation) was significantly higher in the non-cross-linked porcine collagen scaffold compared with PRF-M and fibrin matrix based on platelet-rich plasma. There were no significant differences found in the results of the live/dead assay., Conclusions: Significant differences in the response of human MSCs to biologic scaffolds existed. MSC adhesion, proliferation, and scaffold morphology evaluated by histologic analysis and electron microscopy varied throughout the evaluated types of scaffolds. Non-cross-linked porcine collagen scaffolds showed superior results for cell adhesion and proliferation, as well as on histologic evaluation., Clinical Relevance: This study enables the clinician and scientist to choose scaffold materials according to their specific interaction with MSCs., (Copyright © 2014 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Food choices can involve a moral element of which healthy eating has come to play a major part in recent years. This research aimed to explore the moral space of food for older adults in order to understand how they conceptualise and negotiate various moral demands in the context of their general food lives. In-depth interviews on the lived experience of food and eating were conducted with a purposive sample of 50 adults aged 50-70, who varied by dietary quality and health status. An inductive thematic analysis was carried out. Three major themes representing aspects of the "moral space of food" were identified. This moral space was influenced by old religious and secular moralities which have become intertwined with new moralities of "healthism", a trend towards encouraging personal responsibility for health. Participants sought to maintain moral congruence by keeping their behaviour within moral boundaries through balance and moderation. Some resisted immoral positioning by highlighting their own autonomy or by challenging healthist ideology. A fundamental tension exists between the concept of healthy eating as desirable to remain a moral person while simultaneously being equated with sacrifice of pleasure and enjoyment. Healthist ideology perpetuates this tension, problematising enjoyment of food and bodies of those outside of the "norm". Attempting to address negative moralistic undertones of healthy eating messages may help to engage public interest in nutrition., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This research considers the processes involved in the formation of attitudes by citizens on potentially contentious novel food technologies (NFTs). Observations of one-to-one deliberative discourses between food scientists and citizens, during which they discussed these technologies, form the basis of this enquiry. This approach enables an exploration of how individuals construct meaning around as well as interpret information about the technologies. Thematic analysis identifies key features that provide the frameworks for citizens' evaluations. How individuals make sense of these technologies is shaped by their beliefs, values and personal characteristics; their perceptions of power and control over the development and sale of NFT related products; and, the extent to which these products are relevant to their personal lives. Internal negotiations between these influences are evident, and evaluations are based on the relative importance of each influence to the individual. Internal conflicts and tensions are associated with citizens' evolving evaluative processes, which may in turn present as attitude ambivalence and instability. Many challenges are linked with engaging with the general public about these technologies, as levels of knowledge, understanding and interest vary., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: Biologics may help to optimize the healing environment after rotator cuff repair. Mesenchymal stem cells (MSCs) may have the potential to regenerate a physiological enthesis, thereby improving healing at the repair site after rotator cuff repair., Methods: The PubMed database was searched in May 2013. Only in vivo and in vitro studies reporting on stem cell use in the rotator cuff of humans or animals were included. Exclusion criteria consisted of the following: Level V evidence, systematic reviews, and studies reporting preliminary results., Results: This query resulted in 141 citations. Of these, 90 were excluded based on the title of the study. A final group of 17 studies was included in this review (9 in vivo animal studies, 5 in vitro human studies, 1 in vitro animal study, 1 study reporting in vitro human and in vivo animal results, and 1 study reporting on clinical outcomes of human patients)., Conclusions: The current literature regarding therapeutic use of MSCs in shoulder surgery is limited. Although in vivo animal studies have shown some promising approaches to enhance tendon-to-bone healing, the use of MSCs for shoulder surgery should still be regarded as an experimental technique. Further basic and clinical research is needed until a procedure can be defined for the routine use of these cells in shoulder surgery., (Copyright © 2013 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Objective: The purpose of this study was to compare CT with conventional and simulated reduced-tube current in the evaluation for acute appendicitis in children., Materials and Methods: Validated noise-addition (tube current-reduction) software was used to create 50% and 75% tube current reductions in 60 CT examinations performed for suspected appendicitis, resulting in 180 image sets. Three blinded pediatric radiologists scored the randomized studies for the following factors: presence of the normal appendix or appendicitis (5-point scale; 1=definitely absent and 5=definitely present), presence of alternate diagnoses, and overall image quality (1=nondiagnostic and 5=excellent). Truth was defined by the interpretation of the conventional examination., Results: For conventional examinations, the total number of reviews (60 cases×3 readers=180) in which the normal appendix was identified was 120 of 180 (66.7%), compared with 108 of 180 (60%) in the 50% (p=0.19) and 91 of 180 (50.6%) in the 75% (p=0.002) tube current-reduction groups. Appendicitis was identified in a total of 39 of 180 (21.7%), 38 of 180 (21.1%), and 37 of 180 (20.6%) examinations, respectively (p>0.05). This translates to sensitivities of 97% and 95% for the 50% and 75% tube current-reduction groups, respectively. Alternate diagnoses were detected in 14%, 16%, and 13% of scans, respectively. Compared with conventional-tube current examinations, reader confidence and assessment of image quality were significantly decreased for both tube current-reduction groups., Conclusion: Simulated tube current-reduction technology provides for systematic evaluation of diagnostic thresholds. Application of this technology in the setting of suspected appendicitis shows that tube current can be reduced by at least 50% without significantly affecting diagnostic quality, despite a decrease in reader confidence and assessment of image quality.

Purpose: The purpose of this study was to evaluate the effect on cell viability of the isolated and combined use of allogeneic platelet-rich plasma (PRP) and ketorolac tromethamine on human chondrocytes and tenocytes in a highly controlled in vitro environment., Methods: PRP was produced from 8 subjects. Human chondrocytes (Lonza, Hopkinton, MA) and tenocytes isolated from samples of the long head of the biceps tendons were treated in culture with PRP, ketorolac tromethamine, and methylprednisolone, both alone and in combination. Control samples were treated in media containing 2% or 10% fetal bovine serum (FBS). Cells were exposed for 1 hour. Luminescence assays were obtained to examine cell viability after 24 hours and long-term effects on cell viability after 120 hours. Radioactive thymidine assay was used to measure proliferation after 120 hours., Results: For chondrocytes, cell viability (120 hours) increased significantly with the treatment of PRP alone (43,949 ± 28,104 cells; P < .001) and with the combination of ketorolac tromethamine and PRP (43,276 ± 31,208; P < .001), compared with the 2% FBS group (7,397 ± 470). Cell viability decreased significantly after exposure to methylprednisolone (1,323 ± 776; P < .001) and its combination with PRP (4,381 ± 5,116; p < .001). For tenocytes, cell viability (120 hours) was significantly higher with the treatment of PRP (61,287 ± 23,273; P < .001) and the combined treatment of ketorolac tromethamine and PRP (52,025 ± 17,307; P < .001), compared with the 2% FBS group (23,042 ± 2,973). Cell viability decreased significantly after exposure to methylprednisolone (3,934 ± 1,791; P = .001) and its combination with PRP (5,201 ± 2,834; P = .003), compared with 2% FBS., Conclusions: Tendon and cartilage cells showed increased cell viability after an exposure to allogeneic PRP and ketorolac tromethamine. Exposure to methylprednisolone alone decreased cell viability, and addition of PRP could partially reverse this negative effect., Clinical Relevance: Intra-articular injections of pain-modifying or anti-inflammatory drugs are routinely given in orthopaedic practice. Among the many agents available for intra-articular injection, corticosteroids and local anesthetics are the most common in clinical practice. Potential detrimental side effects of intra-articular injections of corticosteroids and local anesthetics have prompted investigation into alternative treatment options such as combinations of PRP and ketorolac tromethamine. In vitro evaluation of their effect on cell viability might build a basis for further translational research and clinical application., (Copyright © 2013 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Purpose: To assess molecular and histologic differences between the proximal (intra-articular) and distal (extra-articular) portions of the long head of the biceps (LHB) tendon in 3 different disease states (biceps instability, tendinosis, and degenerative joint disease [DJD]) compared with a healthy tendon (fresh frozen)., Methods: We used 32 LHB tendons of patients undergoing tenodesis (mean age, 54.7 ± 10.1 years) and 9 harvested tissue donors. Tendons were divided according to 4 diagnostic groups: (1) biceps instability, (2) tendinosis, (3) DJD, and (4) normal control. After sectioning, tendons were fixed in formalin and stained with H&E and alcian blue for histologic analysis. Measurements of collagen organization by use of polarized light microscopy was then performed, and protein expression for type I and type III collagen, tenascin C, and decorin was determined., Results: There were no statistical differences found for protein expression of type I or type III collagen, tenascin C, or decorin. The proximal and distal regions of the tendons had statistically significant differences in alcian blue staining, with the proximal portion containing a higher amount of proteoglycan (instability, P = .001; tendinosis, P = .005; DJD, P = .008; control, P = .011). When compared with the nonpathologic control tendons, a significant increase in alcian blue staining for the proximal region was seen in all 3 groups. Total polarized light analysis showed that the distal tendon had a significantly higher intensity (organization) compared with the proximal tendon (P < .001); this was also seen in all of the diagnostic groups (instability, P = .010; tendinosis, P = .013; DJD, P = .07; control, P = .028)., Conclusions: This study showed a greater degree of degeneration of the proximal (intra-articular) regions of the LHB tendon when compared with the distal regions in all pathologic groups. However, no major differences at the cellular level were found among groups., Clinical Relevance: The pathomechanisms of the various forms of known LHB diagnoses are not yet fully understood and basic science studies may help in understanding their etiology and therefore optimizing treatment options., (Copyright © 2013 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to quantify the extent of the anti-inflammatory effect of platelet-rich plasma (PRP) in a controlled in vitro environment., Methods: Through the stimulation of human umbilical vein endothelial cells with inflammatory cytokines (tumor necrosis factor α and interferon γ), cell adhesion molecule expression (E-selectin, vascular cell adhesion molecule, and human leukocyte antigen DR) and PRP's anti-inflammatory effect can be measured. PRP was produced from 3 individuals using a single-spin (PRPLP) process. Treatment groups include negative (unstimulated) controls, positive (stimulated) controls, ketorolac tromethamine, methylprednisolone, PRP, ketorolac-PRP, and methylprednisolone-PRP. A fluorescence assay of the cellular inflammation markers was measured by the BioTek Synergy HT plate reader (BioTek Instruments, Winooski, VT) at 0, 1, 2, and 5 days., Results: At days 2 and 5, methylprednisolone treatment showed a 2.1- to 5.8-fold reduction (P < .05) in inflammation markers over PRP. In addition, PRP and ketorolac showed a 1.4- to 2.5-fold reduction (P < .05) in cellular inflammation markers over the control. There was no statistically significant difference between ketorolac and PRP., Conclusions: Although PRP and ketorolac reduced cellular inflammation markers (E-selectin, vascular cell adhesion molecule, and human leukocyte antigen DR) compared with control, neither caused as great a reduction as methylprednisolone., Clinical Relevance: Although PRP and ketorolac did not produce as significant a reduction in cellular inflammation markers as methylprednisolone, they reduced cellular inflammation compared with the control. These agents may have clinical application as injectable anti-inflammatory medications., (Copyright © 2013 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Purpose: The aim of this study was to examine the relations between age, gender, and number of viable mesenchymal stem cells (MSCs) in concentrated bone marrow (BM) obtained from the proximal humerus and distal femur during arthroscopic surgery., Methods: BM was aspirated from either the proximal humerus (n = 55) or distal femur (n = 29) during arthroscopic surgery in 84 patients (51.3 ± 11.6 years). MSCs were obtained from fractionated bone marrow after a 5-minute spin at 1,500 rpm. Volume of BM and number of nucleated cells (NCs) were calculated, and samples were cultured for 6 days, after which point colony-forming units (CFUs) were quantified and fluorescence-activated cell sorting (FACS) analysis was performed. Simple linear regression was used to explore relations between age, gender, volume of aspirated BM, and MSCs per milliliter., Results: BM aspirations yielded a mean quantity of 22.6 ± 12.3 mL. After centrifugation, 30.0 ± 16.7 × 10(6) nucleated cells/mL of concentrated BM were harvested. The proximal humerus provided 38.7 ± 52.6 × 10(6), and the distal femur, 25.9 ± 14.3 × 10(6), for an overall 766.3 ± 545.3 MSCs/mL of concentrated BM (proximal humerus: 883.9 ± 577.6, distal femur: 551.3 ± 408.1). Values did not significantly differ by age, gender, or donor site., Conclusions: Arthroscopic aspiration of bone marrow from the proximal humerus and distal femur is a reproducible technique and yields reliable concentrations of MSCs. The use of an intraoperative concentration method resulted in consistent amounts of MSCs in all clinically relevant age groups without a significant drop of the number of isolated MSCs., Clinical Relevance: Human MSCs derived from concentrated bone marrow aspirate are a promising biological addition that may have practical use in the future of soft tissue augmentation. Arthroscopic techniques for bone marrow aspiration that do not require an additional surgical site for aspiration (e.g., iliac crest) or a second operative procedure may facilitate future use of MSCs in arthroscopic surgery., (Copyright © 2013 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Purpose: Results of in vitro cell models are commonly used to promote new therapies (e.g., platelet-rich plasma), and clinicians have to be aware of the specific limitations of such models. To gain a sufficient and effective cell load, many current in vitro models use cells multiplied through various passages. This is especially important in tendon-like cell (TLC) models, since native tendon tissue is not available unlimited and contains limited amount of tenocytes. The purpose was to determine the occurrence of phenotypic changes following extended monolayer culture of TLCs, according to cell-passage number., Methods: Tendon samples were obtained from 15 healthy patients undergoing biceps tenodesis. Tendons were digested and cultured (monolayer) for six passages. Tendon-specific markers (collagens I and III, decorin, tenascin-C, and tenomodulin) and their histology were analyzed using gene expression and protein content assays. Native cells, the cells cultured and cells passaged one to six times were analyzed at each passage., Results: Gene expression of types I and III collagen of cultured TLCs significantly decreased after two passages. Gene expression of decorin, tenascin-C, and tenomodulin exhibited a trend of decreased gene expression with increased passage. Protein levels of types I and III collagen and decorin decreased after four passages., Conclusions: The significant findings let conclude that tenocyte-like cells obtained from human LHB can be maintained in monolayer culture at low passages, before the signs of phenotypic drift are present. But researchers must be aware of rapid phenotypic drift at higher passage numbers. Therefore, only cells within the first 3 passages should be used as a precaution for in vitro monolayer cell models, and one has to be aware of the phenotypic changes if TLCs passaged multiple times are used. The clinical relevance of this data is that understanding of in vitro TLC models, and their limitations may finally help the clinician to judge the potential of experimental data of new biologic treatment options.

Background: Clinical application of platelet-rich plasma (PRP) in the realm of orthopaedic sports medicine has yielded variable results. Differences in separation methods and variability of the individual may contribute to these variable results., Purpose: To compare the effects of different PRP separation methods on human bone, muscle, and tendon cells in an in vitro model., Study Design: Controlled laboratory study., Methods: Blood collected from 8 participants (mean ± SD age 31.6 ± 10.9 years) was used to obtain PRP preparations. Three different PRP separation methods were used: a single-spin process yielding a lower platelet concentration (PRP(LP)), a single-spin process yielding high platelet and white blood cell concentrations (PRP(HP)), and a double-spin that produces a higher platelet concentration and lower white blood cell concentration (PRP(DS)). Human bone, muscle, and tendon cells obtained from discarded tissue samples during shoulder surgery were placed into culture and treated with the 3 PRP preparations, control media (2% fetal bovine serum [FBS] and 10% FBS), and native blood. Radioactive thymidine assays were obtained to examine cell proliferation, and testing with enzyme-linked immunosorbent assay was used to determine growth factor concentrations., Results: Addition of PRP(LP) to osteocytes, myocytes, and tenocytes significantly increased cell proliferation (P ≤ .05) compared with the controls. Adding PRP(DS) to osteoblasts and tenocytes increased cell proliferation significantly (P ≤ .05), but no significance was shown for its addition to myocytes. The addition of PRP(HP) significantly increased cell proliferation compared with the controls only when added to tenocytes (P ≤ .05). Osteoblasts: Proliferation was significantly increased by addition of PRP(LP) compared with all controls (2% FBS, 10% FBS, native blood) (P ≤ .05). Addition of PRP(DS) led to significantly increased proliferation compared with all controls, native blood, and PRP(HP) (P ≤ .05). Proliferation was significantly less when PRP(HP) was added compared with PRP(DS) (P ≤ .05). Myocytes: Proliferation was significantly increased by addition of PRP(LP) compared with native blood (P ≤ .05). Adding PRP(HP) or PRP(DS) to myocytes showed no significant increase in proliferation compared with the controls or the other separations. Tenocytes: Proliferation was significantly increased by addition of PRP(LP) compared with all controls (2% FBS, 10% FBS, native blood) (P ≤ .05). Addition of PRP(DS) showed a significant increase compared with the controls and native blood. For tenocytes, there was a significant increase (P ≤ .05) seen when PRP(HP) was added compared with the controls and native blood but not compared with the other separations., Conclusion: The primary findings of this study suggest the application of different PRP separations may result in a potential beneficial effect on the clinically relevant target cells in vitro. However, it is unclear which platelet concentration or PRP preparation may be optimal for the treatment of various cell types. In addition, a "more is better" theory for the use of higher platelet concentrations cannot be supported. This study was not intended to prove efficacy but to provide a platform for future research to be built upon., Clinical Relevance: The utilization of different PRP separations may result in a potentially beneficial effect on the clinically relevant target cells in vitro, but it is unclear which platelet concentration or PRP preparation may be optimal for the treatment of various cell types.

Background: Rotator cuff reconstructions may be improved by adding growth factors, cells, or other biologic factors into the repair zone. This usually requires a biological carrier (scaffold) to be integrated into the construct and placed in the area of tendon-to-bone healing. This needs to be done without affecting the constructs mechanics. Hypothesis/, Purpose: The hypothesis was that scaffold placement, as an interposition, has no adverse effects on biomechanical properties of double-row rotator cuff repair. The purpose of this study was to examine the effect of scaffold interposition on the initial strength of rotator cuff repairs., Study Design: Controlled laboratory study., Methods: Twenty-five fresh-frozen shoulders (mean age: 65.5 ± 8.9 years) were randomly assigned to 5 groups. Groups were chosen to represent a broad spectrum of commonly used scaffold types: (1) double-row repair without augmentation, (2) double-row repair with interposition of a fibrin clot (Viscogel), (3) double-row repair with interposition of a collagen scaffold (Mucograft) between tendon and bone, (4) double-row repair with interposition of human dermis patch (ArthroFlex) between tendon and bone, and (5) double-row repair with human dermis patch (ArthroFlex) placed on top of the repair. Cyclic loading to measure displacement was performed to 3000 cycles at 1 Hz with an applied 10- to 100-N load. The ultimate load to failure was determined at a rate of 31 mm/min., Results: There were no significant differences in mean displacement under cyclic loading, slope, or energy absorbed to failure between all groups (P = .128, P = .981, P = .105). Ultimate load to failure of repairs that used the collagen patch as an interposition (573.3 ± 75.6 N) and a dermis patch on top of the reconstruction (575.8 ± 22.6 N) was higher compared with the repair without a scaffold (348.9 ± 98.8 N; P = .018 and P = .025). No significant differences were found for repairs with the fibrin clot as an interposition (426.9 ± 103.6 N) and the decellularized dermis patch as an interposition (469.9 ± 148.6 N; P = .73 and P = .35)., Conclusion: Scaffold augmentation did not adversely affect the zero time strength of the tested standard double-row rotator cuff repairs. An increased ultimate load to failure was observed for 2 of the augmentation methods (collagen patch as an interposition and decellularized dermis patch on top of the reconstruction) compared with the nonaugmented repairs., Clinical Relevance: Scaffolds intended for application of growth factors or cellular components in a repair situation did not adversely jeopardize the stability of the operative construct.

Purpose: The purpose of this study was to investigate the in vitro effects of an arginine-glycine-aspartic acid (RGD) coating on a high-strength nonabsorbable polyester/polyethylene (PE/PEE) suture material commonly used in orthopaedic procedures., Methods: Human bone and tendon specimens were isolated and cultured. The cells were then plated in known densities in the presence of RGD-coated and uncoated PE/PEE suture and allowed to adhere for predetermined time periods. The RGD-coated and uncoated control sutures were then removed and assayed for cell osteoblast and tenocyte adhesion and proliferation., Results: The RGD-modified suture showed a statistically significant increase in both adhesion and proliferation of human tenocytes when compared with uncoated controls (P < .05)., Conclusions: The RGD peptide sequence can be effectively coupled with commercially available PE/PEE suture. RGD-coated suture is able to stimulate the adhesion and proliferation of human tenocyte cells in vitro, as well as withstand standard sterilization and storage conditions. Furthermore, the acid hydrolysis process did not affect the strength of the suture material., Clinical Relevance: RGD-modified suture materials have the potential to create favorable biologic responses when used in common orthopaedic procedures., (Copyright © 2012 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Background: Varying concentrations of blood components in platelet-rich plasma preparations may contribute to the variable results seen in recently published clinical studies. The purposes of this investigation were (1) to quantify the level of platelets, growth factors, red blood cells, and white blood cells in so-called one-step (clinically used commercial devices) and two-step separation systems and (2) to determine the influence of three separate blood draws on the resulting components of platelet-rich plasma., Methods: Three different platelet-rich plasma (PRP) separation methods (on blood samples from eight subjects with a mean age [and standard deviation] of 31.6 ± 10.9 years) were used: two single-spin processes (PRPLP and PRPHP) and a double-spin process (PRPDS) were evaluated for concentrations of platelets, red and white blood cells, and growth factors. Additionally, the effect of three repetitive blood draws on platelet-rich plasma components was evaluated., Results: The content and concentrations of platelets, white blood cells, and growth factors for each method of separation differed significantly. All separation techniques resulted in a significant increase in platelet concentration compared with native blood. Platelet and white blood-cell concentrations of the PRPHP procedure were significantly higher than platelet and white blood-cell concentrations produced by the so-called single-step PRPLP and the so-called two-step PRPDS procedures, although significant differences between PRPLP and PRPDS were not observed. Comparing the results of the three blood draws with regard to the reliability of platelet number and cell counts, wide variations of intra-individual numbers were observed., Conclusions: Single-step procedures are capable of producing sufficient amounts of platelets for clinical usage. Within the evaluated procedures, platelet numbers and numbers of white blood cells differ significantly. The intra-individual results of platelet-rich plasma separations showed wide variations in platelet and cell numbers as well as levels of growth factors regardless of separation method.

Electrospun polycaprolactone nanofiber matrices surface functionalized with poly[(ethyl alanato), (p-methyl phenoxy),] phosphazene were fabricated for the purpose of soft skeletal tissue regeneration. This preliminary study reports the effect of fiber diameter and polyphosphazene surface functionalization on significant scaffold properties such as morphology, surface hydrophilicity, porosity, tensile properties, human mesenchymal stem cell adhesion and proliferation. Six fiber matrices comprised of average fiber diameters in the range of 400-500, 900-1000, 1400-1500, 1900-2000, 2900-3000 and 3900-4000 nm were considered for primary evaluation. After achieving the greatest proliferation while maintaining moderate tensile modulus, matrices in the diameter range of 2900-3000 nm were selected to examine the effect of coating with 1%, 2% and 3% (weight/volume) polyphosphazene solutions. Polyphosphazene functionalization resulted in rougher surfaces that correlated with coating solution concentration. Analytical techniques such as energy dispersive X-ray analysis, Fourier transform infrared spectroscopy, elemental analysis, differential scanning calorimetry, water contact angle goniometry and confocal microscopy confirmed the presence of polyphosphazene and its distribution on the functionalized fiber matrices. Functionalization achieved through 2% polymer solutions did not affect average pore diameter, tensile modulus, suture retention strength or cell proliferation compared to PCL controls. Surface polyphosphazene functionalization significantly improved the matrix hydrophilicity evidenced through decreased water contact angle of PCL matrices from 130 degrees to 97 degrees. Further, enhanced total protein synthesis by cells during in vitro culture was seen on 2% PPHOS functionalized matrices over controls. Improving PCL matrix hydrophilicity via proposed surface functionalization may be an efficient method to improve cell-PCL matrix interactions.

Purpose: (1) To safely obtain bone marrow aspirates from the distal femur during arthroscopic knee surgery, (2) to purify and efficiently concentrate connective tissue progenitor cells (CTPs) in the operating room (OR), and (3) to confirm that these are CTPs through their ability to differentiate into bone cells., Methods: Bone marrow aspirates were harvested from the distal femur during arthroscopic knee surgery in 26 patients. Twenty-five matched control subjects were selected to evaluate for increased incidence of complications. CTPs were isolated using a rapid method designed for use in the OR compared with 2 accepted methods. Cytochemical and molecular analysis was used to assess osteogenic potential., Results: Osteogenic potential of the CTPs was confirmed by reverse transcription polymerase chain reaction analysis and cellular staining. Bone marrow was successfully aspirated in 25 cases, with 3 incidences of stiffness in the aspirate group compared with 2 in the control group, 1 incidence of a wound irregularity in the aspirate group compared with 1 in the control group, and 3 incidences of hemarthrosis/persistent effusion in the aspirate group compared with 1 in the control group. The rate of complications for the aspirate group was 36% compared with 25% in the control group., Conclusions: Our intention was to develop a technique for extracting and purifying bone marrow so that the orthopaedic surgeon would have a simple, safe, and efficient process by which to isolate CTPs during arthroscopic knee surgery. This method of aspiration did not lead to a significant increase in complications. Further bone marrow aspirate was successfully purified in the OR, with only a slight increase in surgery time, and resulted in a fractionated layer rich with CTPs. These cells showed osteogenic potential, as evidenced by their osteoblastic differentiation. These CTPs may have future use in enhancing the incorporation of the graft into the bone., Level of Evidence: Level III, matched case-control study., (Copyright © 2012 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Purpose: To evaluate the response of human tenocytes in culture to 7 commercially available extracellular matrix (ECM) patches., Methods: Four samples of each ECM were incubated in human tenocyte cultures by use of standard methods. Cell adhesion, cell proliferation, and cellular production of type I and type III collagen, decorin, and scleraxis were measured for each sample according to established experimental methods. Histologic samples were examined to measure the migration of the tenocytes into the ECM., Results: Tenocytes adhered more to samples of layered submucosal pig intestine than the 6 other ECM materials (P < .002). Tenocytes proliferated more and produced more matrix proteins when cultured on ECM derived from unaltered dermal specimens of human or porcine origin (P < .001). Cells were not seen to have migrated into the matrix of any ECM sample., Conclusions: Human tenocytes reacted most favorably to dermal ECM samples that were not chemically cross-linked by the manufacturer. Less favorable responses of the human cells were seen when cultured with equine or synthetic ECM, which showed favorable biologic responses in nonhuman models. Cellular migration into the matrix of the ECM is a complex process and cannot be replicated in this model entirely., Clinical Relevance: The results of this study suggest that dermal ECM may more favorably react with human tendon tissue than ECM of other origins. This may have great relevance as research continues in the field of augmenting surgical soft-tissue repair., (2010 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Background: Bone-to-tendon healing in the shoulder can be unpredictable. Biologic augmentation, through the implementation of adult mesenchymal stem cells, may improve this healing process., Purpose: The purpose of this study was to (1) arthroscopically obtain bone marrow aspirates from the proximal humerus during rotator cuff repair, (2) purify and concentrate the connective tissue progenitor cells (CTPs) in the operating room efficiently, and (3)confirm these are stem cells through their ability to differentiate into bone cells. We hypothesize that CTPs can be quickly and efficiently isolated from bone marrow during arthroscopic surgery and that these cells are capable of osteogenesis., Study Design: Cohort study; Level of evidence, 3; and Descriptive laboratory study., Methods: Bone marrow aspirates were harvested through the anchor tunnel of the humeral head during arthroscopic rotator cuff repair in 23 patients. Twenty-three matched controls were selected from a clinical registry to evaluate for increased incidence of complication. Connective tissue progenitor cells were isolated using 2 accepted methods and compared with a novel, rapid method designed for use in the operating room. Osteogenic potential was assessed by cytochemical and molecular analysis., Results: Reverse transcription polymerase chain reaction analysis and cellular staining confirmed the osteogenic potential of these CTPs. There was no statistical significant difference in the Single Assessment Numeric Evaluation score (aspirate, 86.3 +/- 10.5; control, 83.6 +/- 15.1; P = .54), range of motion measures (postoperative external rotation: aspirate, 65.0 degrees +/- 20.4 degrees ; control, 62.5 degrees +/- 17.1 degrees ; P = .67; postoperative forward elevation: aspirate, 163.0 degrees +/- 30.6 degrees ; control, 145.7 degrees +/- 41.4 degrees ; P = .12), or postoperative strength measures between groups (median, 5; range, 4-5 in the aspirate group compared with median, 5; range, 4-5 in the control group; P > .05)., Conclusion: Connective tissue progenitor cells can be safely and efficiently aspirated from the proximal humerus using the anchor tunnel created during arthroscopic rotator cuff surgery. These cells may play an important role in cell-based therapies involving rotator cuff repair., Clinical Relevance: We have established a reliable, reproducible protocol for isolating CTPs in the operating room. These cells may have the potential to enhance the healing process after rotator cuff repair.

Surgical interference screws and suture anchors for attaching soft tissue, such as ligaments and tendons, to bone are routinely used in arthroscopic surgery and sports medicine. Interference screw fixation provides a press fit between bone, graft/tendon, and screw and is frequently used to attach replacement ligaments in tunnels drilled for anterior and posterior cruciate ligament reconstruction. Suture anchors are used in surgical procedures wherein it is necessary for a surgeon to attach (tie) tissue to the surface of the bone, for example, during joint reconstruction and ligament repair or replacement. The composition of these implants ranges from metals to polymers and composites. Typically, because of the relatively large amount of torque that must be applied during insertion, these screws are constructed from metal. However, interference screws and suture anchors have also been constructed from bioabsorbable polymers and composites. The ideal material would (1) provide adequate mechanical fixation, (2) completely degrade once no longer needed, and (3) be completely replaced by bone. Because no material has been shown to be superior for all applications, the surgeon must weigh the advantages and disadvantages of each to evaluate the optimum material for a given application and patient. The purpose of this article is to present a comprehensive review of the commercially available interference screws and suture anchors, with an emphasis on implant composition, interaction, and design. This article provides the orthopaedic surgeon with a background on biomaterials, specifically those used in interference screws and suture anchors. Because there is no material that is perfect for all surgical situations, this review can be used to make educated decisions on a case-by-case basis., (Published by Elsevier Inc.)

Tendinopathy is a vexing clinical problem as its onset and development is often asymptomatic and unrecognized until tendon rupture. While extensively studied in the rotator cuff, Achilles, and patellar tendons, no study to date has examined the histological and molecular characteristics of the tendinopathic biceps long-head (LHB). The anatomy of the LHB is unique in that it comprises intra- and extra-articular portions, each exposed to differing loading patterns. Eleven LHBs post-tenotomy were sectioned, fixed in formalin, and stained (H and E; Alcian Blue), and gross structural organization of collagen measured using polarized light microscopy. Protein expression of intra- and extra-articular portions of the tenotomized biceps for IGF-I, collagen III, and MMP-1, -2, -3, and -13 was determined with Western blot analyses. The intra-articular LHB exhibited significantly greater histological evidence of tendinopathy inclusive of increased proteoglycan (p < 0.05) and decreased organization as measured by polarized light microscopy (p < 0.01). The intra-articular LHB also had significantly increased expression of collagen type III (p < 0.01) and of MMP-1 and 3 (p < 0.01, p < 0.05 respectively). No significant differences were found for IGF-I or for MMP-2 and -13. The intra-articular LHB exhibited histological characteristics of tendinopathy. Protein expression of the intra-articular LHB did not universally display signs of tendinopathy in comparison to the extra-articular portion of the tendon.

Tendon to bone integration after rotator cuff repair is not a reproducible process. During repair, bioabsorbable and nonabsorbable suture material is universally used to facilitate the procedure. Improving the biological architecture of inert suture might aid in overall tendon to bone healing. The objective of our study is to enhance the bone to tendon union by absorbing type I collagen onto high strength nonabsorbable polyester/polyethylene suture commonly used in rotator cuff surgery. Our purpose was to evaluate the tendon and bone cellular response to this novel coated suture compared to uncoated suture. Primary human osteoblasts (HOBs) and tenocytes were plated onto polyester/polyethylene suture that was either uncoated or coated with type I bovine collagen. Cell adhesion to the sutures was assayed at 24 hours. Proliferation was determined at 48 hours by measuring [3H]- Thymidine incorporation in cells attached to the sutures. At 24 and 48 hours, respectively, cells grown on the collagen-coated suture showed a significantly greater response measured by adhesion and proliferation than cells grown on uncoated suture. At five days of culture, alkaline phosphatase activity and protein synthesis was significantly greater on the collagen-coated suture compared to uncoated. Collagen-coated polyester/polyethylene suture appears to stimulate adhesion, proliferation alkaline phosphatase, and protein synthesis more than uncoated sutures, and therefore may aid in the tendon to bone incorporation process critical to rotator cuff repair.

Apoptosis is a process important for the development and homeostasis of self-renewing tissues, including bone. However, little is known about the function of Bcl-2, a key player of apoptosis, in the regulation of osteoblast activity. Ex vivo cultures of osteoblasts from Col2.3Bcl-2 mice, in which human Bcl-2 was targeted to bone by the 2.3 kb fragment of the type I collagen promoter, were used to study the effect of Bcl-2 in osteoblasts. During 35 days of culture, hBcl-2 expression increased without any effect on endogenous mouse Bcl-2 and Bax expression. Adhesion of transgenic (TG) osteoblasts was twofold more than that of wild-type (WT) cells, with significantly higher expression of integrins alpha(1), alpha(2), and alpha(5) but similar levels of alpha(v) and beta(1) relative to WT cells. Proliferation of osteoblasts was not affected. Overexpression of hBcl-2 promoted the differentiation of osteoblasts, as shown by increased message levels of alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin in the TG compared to WT cells throughout the culture period. The two transcription factors essential for osteoblast differentiation, core binding factor alpha 1 (Cbfa-1) and osterix, had significantly higher expression in TG than WT cells during the early culture period. ss-Catenin, a central player in the canonical Wnt pathway, also had higher expression in TG than WT cultures. Mineralization was significantly decreased in TG cultures, with less osteoblast apoptosis, compared to WT. Thus, Bcl-2 seems to have multiple roles in modulating osteoblast activities.

Tendon reconstruction surgery often requires healing of the tendon to bone. The development of a more rapid and strong interaction at the tendon to bone interface would be invaluable to patients having orthopaedic surgery. Therefore, our rationale was to modify sutures so that they would be anabolic for tendon to bone healing. It has been shown that silk stimulates bone formation in osteoblast cultures. In the current study, we tested the ability of silk and silk-RGD (arginine-glycine-aspartic acid) to stimulate human tenocyte adhesion, proliferation, and differentiation. A 1.3-fold increase in tenocyte adhesion was found on silk-RGD compared with tissue culture plastic. By 72 hours, proliferation had increased on all substrates but was particularly enhanced on silk-RGD compared with the control. At 6 weeks, Northern blot analysis of decorin and Type I collagen mRNA levels showed a 2-3-fold increase in message levels on silk-RGD and silk compared with tissue culture plastic. The data suggest cultured human tenocytes adhere, proliferate, and differentiate on silk and silk-RGD substrates. A suture material, such as silk, decorated with RGD, may have the potential to facilitate tendon-bone healing with widespread applications in tendon reconstruction surgery.

Unlabelled: Transgenic mice (Col2.3Bcl-2) with osteoblast-targeted human Bcl-2 expression were established. Phenotypically, these mice were smaller than their wildtype littermates and showed differential effects of the transgene on bone parameters and osteoblast activity dependent on sex. The net effect was an abrogation of sex differences normally observed in wildtype mice and an inhibition of bone loss with age. Ex vivo osteoblast cultures showed that the transgene had no effect on osteoblast proliferation, but decreased bone formation. Estrogen was shown to stimulate endogenous Bcl-2 message levels. These studies suggest a link between Bcl-2 and sex regulation of bone development and age-related bone loss., Introduction: Whereas Bcl-2 has been shown to be an important regulator of apoptosis in development, differentiation, and disease, its role in bone homeostasis and development is not well understood. We have previously showed that the induction of glucocorticoid-induced apoptosis occurred through a dose-dependent decrease in Bcl-2. Estrogen prevented glucocorticoid-induced osteoblast apoptosis in vivo and in vitro by preventing the decrease in Bcl-2 in osteoblasts. Therefore, Bcl-2 may be an important regulator of bone growth through mechanisms that control osteoblast longevity and function., Materials and Methods: Col2.3Bcl-2 mice were developed carrying a 2.3-kb region of the type I collagen promoter driving 1.8 kb of human Bcl-2 (hBcl-2). Tissue specific expression of hBcl-2 in immunoassays validated the transgenic animal model. Histomorphometry and DXA were performed. Proliferation, mineralization, and glucocorticoid-induced apoptosis were examined in ex vivo cultures of osteoblasts. The effect of estrogen on mouse Bcl-2 in ex vivo osteoblast cultures was assayed by RT-PCR and Q-PCR., Results and Conclusions: Two Col2.3Bcl-2 (tg/+) founder lines were established and appeared normal except that they were smaller than their nontransgenic wildtype (+/+) littermates at 1, 2, and 6 months of age, with the greatest differences at 2 months. Immunohistochemistry showed hBcl-2 in osteoblasts at the growth plate and cortical surfaces. Nontransgenic littermates were negative. Western blots revealed hBcl-2 only in type I collagen-expressing tissues. Histomorphometry of 2-month-old mice showed a significant decrease in tg/+ calvaria width with no significant differences in femoral trabecular area or cortical width compared with +/+. However, tg/+ males had significantly more trabecular bone than tg/+ females. Female +/+ mice showed increased bone turnover with elevated osteoblast and osteoclast parameters compared with +/+ males. Col2.3Bcl-2 mice did not show such significant differences between sexes. Male tg/+ mice had a 76.5 +/- 1.5% increase in ObS/BS with no significant differences in bone formation rate (BFR) or mineral apposition rate (MAR) compared with male +/+ mice. Transgenic females had a significant 48.4 +/- 0.1% and 20.1 +/- 5.8% decrease in BFR and MAR, respectively, compared with +/+ females. Osteoclast and osteocyte parameters were unchanged. By 6 months, femurs from female and male +/+ mice had lost a significant amount of their percent of trabecular bone compared with 2-month-old mice. There was little to no change in femoral bone in the tg/+ mice with age. Ex vivo cultures of osteoblasts from +/+ and Col2.3Bcl-2 mice showed a decrease in mineralization, no effect on proliferation, and an inhibition of glucocorticoid-induced apoptosis in Col2.3Bcl-2 cultures. Estrogen was shown to increase mouse Bcl-2 transcript levels in osteoblast cultures of wildtype mice, supporting a role for Bcl-2 in the sex-related differences in bone phenotype regulated by estrogen. Therefore, Bcl-2 differentially affected bone phenotype in male and female transgenic mice, altered bone cell activity associated with sex-related differences, and decreased bone formation, suggesting that apoptosis is necessary for mineralization. In addition, Bcl-2 targeted to mature osteoblasts seemed to delay bone development, producing a smaller transgenic mouse compared with wildtype littermates. These studies suggest that expression of Bcl-2 in osteoblasts is important in regulating bone mass in development and in the normal aging process of bone.

Similar to chick, mouse, and Xenopus, a number of intercellular signaling pathways are required for zebrafish segmentation. However, the spatial scales over which these signaling pathways operate in zebrafish remain largely unknown. During zebrafish segmentation, waves of her1 transcription (a) initiate within the tailbud region, (b) propagate anteriorly through presomitic mesoderm (PSM), and (c) terminate in the anlage of newly-forming somites. These observations raise the question of whether the tailbud region serves as a "pacemaker" or "organizing center" for the initiation of propagating her1 expression waves. Microsurgical manipulations reveal that the anteriorly waves of her1 transcription are not perturbed by removal of the zebrafish tailbud. Furthermore, expression patterns of deltaD, paraxial protocadherin C (papc), and myoD within recently formed somites also appear to be relatively unperturbed by either removal of the tailbud, or by removal of lateral plate mesoderm. Although dynamic gene networks rapidly specify mesenchymal or epithelial cellular identities within forming somites, this specification is plastic. Time-lapse analysis has shown that the cellular progeny of mitotically-active epithelial border cells within newly formed somites can adopt different cellular identities than their precursor cells. Overall, these results indicate that sustained long-range intercellular communication with the tailbud, anterior somites, or lateral plate mesoderm is not necessary for segmentation or somitogenesis to proceed within the PSM. The basic segmentation and somitogenesis processes in zebrafish presomitic mesoderm appear to be largely regionally autonomous and governed by local morphogenetic cell behaviors.

The ability of insulin-like growth factor II (IGF-II) to modulate apoptosis was studied in murine osteoblasts. At 72 h of culture, 0.01, 0.1, and 1.0 nM IGF-II produced a dose-dependent increase in apoptosis assayed by TdT-mediated dUTP-biotin nick end labeling (TUNEL) and confirmed with acridine orange-ethidium bromide staining. A maximal increase of 5.0-fold above control was found with 1 nM IGF-II. A time course of treatment with 0.1 nM IGF-II demonstrated a significant increase in apoptosis compared to vehicle-treated cells by 48 h. IGF-II-induced apoptosis could not be inhibited by a blocking antibody to the IGF-I receptor. Human osteoblast cultures demonstrated a similar dose-dependent increase in apoptosis with IGF-II. No significant effect of IGF-II was found on proliferation in murine osteoblast cultures. Western blot analysis demonstrated that IGF-II decreased Bcl-2 protein levels, but not Bax, resulting in a significant reduction in the Bcl-2/Bax ratio. To determine if overexpression of Bcl-2 could block IGF-II-induced apoptosis, osteoblasts were isolated from a transgenic mouse that overexpresses human Bcl-2 in bone through a construct utilizing the 2.3 kb promoter region of the Type I collagen gene linked to a 1.8 kb region of human Bcl-2 (Col2.3Bcl-2). At 72 h, IGF-II significantly increased apoptosis in a dose-dependent manner in osteoblast cultures from the control littermates. In osteoblasts from Col2.3Bcl-2 mice, no significant effect on apoptosis was found with 0.01, 0.1, or 1.0 nM IGF-II. Western blot analysis of Bcl-2 and Bax levels demonstrated a transient decrease in the Bcl-2/Bax ratio at 24 h with no decrease in the ratio at 48 or 72 h. Thus, IGF-II appears to promote osteoblast apoptosis, and overexpression of Bcl-2 is able to block IGF-II-induced apoptosis.

Scientists who study zebrafish currently have an acute need to increase the rate of visual data exchange within their international community. Although the Internet has provided a revolutionary transformation of information exchange, the Internet is at present unable to serve as a vehicle for the efficient exchange of massive amounts of visual information. Much like an overburdened public water system, the Internet has inherent limits to the services it can provide. It is possible, however, for zebrafishologists to develop and use virtual intranets (such as the approach we outlined in this chapter) to adapt to the growing informatics need of our expanding research community. We need to assess qualitatively the economics of visual bioinformatics in our research community and evaluate the benefit:investment ratio of our collective information-sharing activities. The development of the World Wide Web started in the early 1990s by particle physicists who needed to rapidly exchange visual information within their collaborations. However, because of current limitations in information bandwidth, the World Wide Web cannot be used to easily exchange gigabytes of visual information. The Zebrafish DVD Exchange Project is aimed at by-passing these limitations. Scientists are curiosity-driven tool makers as well as curiosity-driven tool users. We have the capacity to assimilate new tools, as well as to develop new innovations, to serve our collective research needs. As a proactive research community, we need to create new data transfer methodologies (e.g., the Zebrafish DVD Exchange Project) to stay ahead of our bioinformatics needs.

Silks are being reassessed as biomaterial scaffolds due to their unique mechanical properties, opportunities for genetic tailoring of structure and thus function, and recent studies clarifying biocompatibility. We report on the covalent decoration of silk films with integrin recognition sequences (RGD) as well as parathyroid hormone (PTH, 1-34 amino acids) and a modified PTH 1-34 (mPTH) involved in the induction of bone formation. Osteoblast-like cell (Saos-2) responses to the decorated silk films indicate that the proteins serve as suitable bone-inducing matrices. Osteoblast-like cell adhesion was significantly increased on RGD and PTH compared to plastic, mPTH, and the control peptide RAD. At 2 weeks of culture, message levels of alkaline phosphatase were similar on all substrates, but by 4 weeks, alkaline phosphatase mRNA was greatest on RGD. At 2 weeks of culture, alpha 1(I) procollagen mRNA was elevated on silk, RGD, RAD, and PTH, and hardly detectable on mPTH and plastic. However, by 4 weeks RGD demonstrated the highest level compared to the other substrates. Osteocalcin message levels detected by RT-PCR were greatest on RGD at both time points. Calcification was also significantly elevated on RGD compared to the other substrates with an increase in number and size of the mineralized nodules in culture. Thus, RGD covalently decorated silk appears to stimulate osteoblast-based mineralization in vitro., (Copyright 2000 John Wiley & Sons, Inc.)

The ability of estrogen to prevent glucocorticoid-induced apoptosis in osteoblasts was studied both in vitro and in vivo. Glucocorticoid treatment for 72 h produced a dose-dependent increase in the number of apoptotic cells, determined by acridine orange/ethidium bromide staining, with a maximal response of 31+/-2% and 26+/-3% with 100 nM corticosterone in primary rat and mouse osteoblasts, respectively. Simultaneous administration of varying concentrations of 17beta-estradiol and 100 nM corticosterone decreased apoptotic osteoblasts in a dose-dependent manner, with a maximal decrease of 70% with 0.01 nM 17beta-estradiol. Terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling also demonstrated glucocorticoid-induced DNA fragmentation that was inhibited by estrogen. Estrogen was shown to inhibit apoptosis induced by lipopolysaccharide treatment. As early as 6 h, Western blots demonstrated a dose-dependent decrease in the Bcl-2/Bax ratio, which reached a minimum of 0.18 in osteoblasts treated with 1000 nM corticosterone for 72 h. This reduction in Bcl-2/Bax was abolished by treating osteoblasts simultaneously with 17beta-estradiol, but not with 17alpha-estradiol. In 7-day-old mice, administration of varying concentrations of dexamethasone for 72 h resulted in a dose-dependent increase in the number of apoptotic osteoblasts as demonstrated by in situ terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling staining of calvaria. A maximum of 22+/-1% apoptotic osteoblasts on the bone surface was found with 1 mg/kg BW dexamethasone compared with 2+/-1% in vehicle-treated mice. Injection of varying concentrations of 17beta-estradiol (0.5-5 mg/kg BW), but not 17alpha-estradiol, with 1 mg/kg dexamethasone produced a dose-dependent decrease in the number of apoptotic osteoblasts to 5+/-1% with 5 mg/kg 17beta-estradiol. Thus, glucocorticoid-induced apoptosis of osteoblasts may be prevented at least in part by 17beta-estradiol.

An in vitro mineralizing cell-implant system was developed to study osteoblast attachment, secretion of extracellular (ECM) matrix proteins and mineralization. Saos-2 cells were plated on Tivanium (Tiv, Ti-6A1-4V), Zimaloy (Zim, Co-Cr-Mo) and glass disks. The cells were cultured in alpha-MEM medium with 10% fetal bovine serum and 50 microg ml(-1) ascorbic acid. The cultures were analyzed for calcification and for mRNA expression for ECM proteins after 1, 2, 4 and 6 weeks. Calcium content was significantly higher in cells on Tiv, less on Zim and least on glass disks. With the addition of 3 mm beta-glycerophosphate (beta-GP), the cell layer was more calcified on Zim than on Tiv and all substrates had three times more calcium than cultures without beta-GP. All subsequent experiments were performed without beta-GP. Phalloidin immunofluorescence microscopy of the actin-based cytoskeleton at 2 weeks demonstrated nodules composed of multilayered, cobblestone-appearing osteoblasts overlying calcified matrix which was stained with calcein. On Tiv, calcified nodules were connected in a trabecular-like pattern while on Zim, calcification was dispersed throughout the cell layer. Northern blots for alkaline phosphatase, bone sialoprotein, osteocalcin and alpha1(I) procollagen mRNAs were performed at different time points. The amount and pattern of calcification as well as the expression of ECM-mRNAs differed on each implant material. The results indicate that Tiv stimulates the production of more ECM proteins and mineralized matrix than Zim or glass in this osteoblast-like cell/implant culture.

The effects of ascorbic acid on collagen synthesis, mineralization, and integrins were investigated in a mineralizing organ culture system derived from 20-day fetal rat parietal bones. A significant dose-dependent decrease in calcification at 96 h was demonstrated with decreasing concentrations of ascorbic acid (100-0 microg/ml). No effect on DNA content, [3H]thymidine incorporation, or dry weight was found in control (100 microg/ml ascorbic acid) bones compared with bones treated with decreased ascorbic acid concentrations (10, 1, and 0 microg/ml). Collagen synthesis, measured by [3H]proline incorporation, and alpha1(I) procollagen messenger RNA levels were also unaffected. However, ascorbic acid produced a dose-dependent decrease in the hydroxyproline content, with a maximal 76.8% decrease in bones without ascorbic acid compared with the control bones with 100 microg/ml ascorbic acid. Light microscopy of the ascorbic acid-deficient bones revealed a disruption of the osteoblast layer with misshapen osteoblasts and a decrease in the osteoid seam. The loss of osteoblast organization was also confirmed by analyzing the integrins for collagen by Northern and Western blot and immunofluorescence microscopy. A dose-dependent decrease in alpha2 and beta1 integrin messenger RNA levels and in alpha1, alpha2, and beta1 protein were found in 96-h bone cultures deficient in ascorbic acid. These integrin subunits mediate the binding of osteoblasts to collagen. Immunofluorescence microscopy also demonstrated a dose-dependent decrease in alpha2 and beta1 staining of the osteoblast layer. However, the protein levels of alpha3 and alpha5 subunits were not affected. No beta5 was detected, whereas only bones cultured without ascorbic acid demonstrated a small decrease in alpha(v) and beta3 protein levels. The alpha3, alpha5, alpha(v), and beta3 subunits are involved in cell binding to extracellular matrix proteins other than collagen. Thus, the integrins for collagen are down-regulated, probably in response to the underhydroxylated collagen fibrils, which causes a disruption of osteoblast organization leading to a decrease in mineralization of bone. Integrin assays for specific extracellular proteins may be useful tools in detecting matrix defects in various metabolic bone diseases.

The initial interaction of the human osteoblast-like cell line Saos-2 with orthopaedic implant materials was analyzed to determine the mechanism by which these cells adhere to implant surfaces. Saos-2 cells were allowed to attach to disks composed of the orthopaedic implant materials Tivanium (Ti6A14V) and Zimaloy (CoCrMo) and to control disks of glass and plastic. Serum had no effect on the number of cells that attached to Tivanium and Zimaloy at 4 or 24 hours but did increase the number of cells that attached to glass at 24 hours. Collagen synthesis was determined by [3H]proline incorporation into collagenase-digestible protein and noncollagen protein. A significant increase of 19% was found for collagen synthesized in cells cultured on Zimaloy for 24 hours compared with glass, with no differences on Tivanium and plastic. However, collagenase-digestible protein and noncollagen protein were increased the most (204 and 198%, respectively) on Tivanium compared with glass. To determine if integrins were involved in cell attachment to implant materials, the peptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), which blocks integrin receptors through the Arg-Gly-Asp sequence, was added to the cells in serum-free medium. This peptide inhibited cell adhesion by 28% on Tivanium and 40% on Zimaloy but had no effect on glass and plastic. The control peptide GRADSP (Gly-Arg-Ala-Asp-Ser-Pro) had no effect on adhesion. Inhibition of protein synthesis and enzymatic removal of surface proteins did not affect the ability of Arg-Gly-Asp peptides to inhibit cell attachment to the implant materials. These results suggest that integrins are able to bind directly to Tivanium and Zimaloy. Western blot analysis of integrin protein demonstrated changes in many integrin subunits, depending on the substrate to which cells attached. In particular, the beta 1 integrin subunit was increased 3.8 to 9.5-fold at 24 hours. To determine specifically which integrins may be involved in adhesion, antibodies to integrins were added. An antibody to the fibronectin receptor, alpha 5 beta 1, significantly inhibited binding of cells to Tivanium by 63% and to Zimaloy by 49% and had no effect on glass. The vitronectin receptor antibody, alpha v beta 3/beta 5, did not alter cell adhesion. In conclusion, osteoblast-like cells appear to be capable of attaching directly to implant materials through integrins. The type of substrate determines which integrins and extracellular matrix proteins are expressed by osteoblasts. These data provide information on how implant materials may affect osteoblast differentiation and bone growth.

Prolonged treatment with glucocorticoids is known to produce osteoporosis, which is characterized by a decrease in bone mass. Therefore, we studied the effect of glucocorticoids on the formation of bone and on the expression of beta 1 integrins in a mineralizing organ culture of fetal rat parietal bone. Integrins are a family of integral membrane glycoproteins that mediate the adhesion of cells to extracellular matrix macromolecules and affect the growth and differentiation of cells. In situ hybridization with a 32P-labeled beta 1 integrin cDNA probe was performed on parietal bone, treated with or without 100-nanomolar corticosterone for ninety-six hours, to localize and assess the levels of beta 1 integrin mRNA quantitatively. Corticosterone decreased beta 1 integrin mRNA in the osteoblast layer but not in the periosteum. Northern blot analysis demonstrated a 62 per cent decrease in the levels of beta 1 integrin mRNA in the osteoblast layer of bone that had been stripped of its periosteum. Immunofluorescence microscopy confirmed these results, as they demonstrated a decrease in the levels of beta 1 integrin protein predominantly in the osteoblast layer. This effect was dependent on the concentration of corticosterone. During ninety-six hours of culture, the calcium content and the dry weight of control parietal bone increased 157 per cent and 57 per cent, respectively. However, treatment of these cultures with 100-nanomolar corticosterone inhibited calcification by 24 per cent. The administration of glucocorticoid had no significant effect on the DNA content or dry weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged glucocorticoid treatment causes osteoporosis in vivo and inhibits bone formation in vitro. We have previously shown that glucocorticoids inhibit calcification and alter osteoblast organization in a mineralizing bone organ culture system. In this study, the effect of glucocorticoids on osteoblast adhesion to bone matrix proteins and integrin expression was examined in primary rat osteoblasts and a transformed rat osteosarcoma-derived cell line ROS 17/2.8. After 24 h of treatment with corticosterone, these cells displayed a concentration-dependent decrease in adhesion to type I collagen and fibronectin. Adhesion was significantly decreased as early as 4 h after glucocorticoid administration. With 100 nM corticosterone treatment for 24 h, inhibition of the adhesion of ROS 17/2.8 cells and primary osteoblasts to fibronectin was 75 +/- 10% and 50 +/- 8%, and inhibition of adhesion to collagen was 31 +/- 10% and 65 +/- 5%, respectively. This effect was specific for osteoblasts, because glucocorticoids did not change the adhesion of fibroblasts. However, glucocorticoids did inhibit the adhesion of all cell types to rat osteonectin. To determine whether the change in osteoblast attachment to collagen and fibronectin was due to an alteration in integrin levels, the plasma membranes of these cells were labeled with [125I]lactoperoxidase, solubilized, and immunoprecipitated with an antibody to beta 1. A 24-h treatment with 100 nM corticosterone caused 80 +/- 2% and 64 +/- 9% decreases in beta 1 levels in primary osteoblasts and ROS 17/2.8 cells, respectively. These results were confirmed with immunofluorescence microscopy, which showed a glucocorticoid-induced decrease in beta 1 staining. Treatment of primary rat osteoblasts and ROS 17/2.8 cells for 72 h with corticosterone also decreased beta 1-integrin messenger RNA levels in a dose-dependent manner. We have demonstrated that the inhibition of integrin expression by glucocorticoids is involved in the decrease in osteoblast adhesion to bone extracellular matrix proteins. These data suggest that integrin modulation may influence osteoblast function and bone formation and, thus, contribute to glucocorticoid-induced osteoporosis.

The effects of corticosterone on fibronectin production, bone growth, and morphology were examined in a mineralizing organ culture system derived from 20-day-old fetal rat parietal bones. During 4 days of culture, 1-1000 nM corticosterone had no significant effect on the increase in dry weight or on DNA content, but 100 and 1000 nM corticosterone did inhibit the increase in calcium content. Light microscopic examination of the 4-day cultures demonstrated a glucocorticoid-induced change in osteoblast shape and organization along the mineralizing front of the bone. A dose-dependent inhibition of fibronectin secretion into the medium was determined by enzyme-linked immunosorbent assay. In control cultures, fibronectin production was 0.105 +/- 0.005 microgram/ml.bone at 24 h and 0.397 +/- 0.037 microgram/ml.bone during the 72- to 96-h interval. The maximal inhibition of fibronectin secretion was 45% at 24 h and 70% at 96 h with 1000 nM corticosterone. Both immunofluorescent visualization of fibronectin staining in the tissue and a Western blot of fibronectin in the tissue showed a decrease in fibronectin levels. At 24 and 96 h, a dose- and time-dependent decrease in fibronectin mRNA transcripts was found. At 24 and 96 h, 1000 nM corticosterone produced a decrease of 42% and 62%, respectively, in fibronectin mRNA levels. Our findings show that glucocorticoids inhibit fibronectin production in developing bone. The decrease in fibronectin synthesis may contribute to altered osteoblast organization and function during bone formation.

The effect of glucocorticoids on bone resorption was examined in a serum-free mineralizing organ culture system derived from 20 day fetal rat parietal bones. Bone resorption was assessed by prelabeling the fetal rats in utero with 45Ca and determining the daily release of 45Ca into the medium of cultured bones. During the first 24 h of treatment a transient stimulation of bone resorption was found; 4.5 +/- 0.3% of the total 45Ca was released into the medium with 1 nM corticosterone and 4.1 +/- 0.2% with 10 nM corticosterone compared to 2.9 +/- 0.2% in control bones. Treatment with 1 and 10 nM dexamethasone for 24 h also showed an increase in 45Ca release compared to control bones. During the same time period 45Ca release was 6.9 +/- 1.4% with 10 nM parathyroid hormone. At later time points 100 and 1000 nM corticosterone inhibited 45Ca release, but 1 and 10 nM corticosterone values were similar to controls. At 24 h the number of osteoclasts per mm2 tissue in bone lacunae was significantly elevated with 1-100 nM corticosterone and 10 nM parathyroid hormone compared to control bones. In control bones 0.10 +/- 0.05 osteoclasts per mm2 of tissue were found, but 0.59 +/- 0.21 osteoclasts per mm2 were seen with 10 nM corticosterone and 1.50 +/- 0.34 with 10 nM parathyroid hormone. An additional assay of bone resorption, the release of lysosomal beta-glucuronidase into the medium was also elevated in glucocorticoid and parathyroid hormone-treated cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

A series of seven hemeproteins, cytochromes P-450LM2, P-450LM4, and P-420LM2, horseradish peroxidase, chloroperoxidase, catalase, and metmyoglobin, as well as hemin were tested for their ability to catalyze a set of five oxidative reactions. These reactions were a typical peroxidative reaction (oxidation of pyrogallol to purpurogallin) and three characteristic P-450 reactions (aliphatic hydroxylation, aromatic hydroxylation, and olefinic epoxidation). In addition, the ability to decarboxylate a peroxyacid was measured. All hemeproteins were able to carry out peroxidation, but three (horseradish peroxidase, chloroperoxidase, and catalase) were much better catalysts than the others. Only the P-450 enzymes were competent catalysts for the hydroxylation and epoxidation reactions. Furthermore, the decarboxylation reaction was strictly limited to the P-450 enzymes, establishing it as a new, unique P-450 activity. Since the decarboxylation of peroxyacids is diagnostic of peroxide homolysis, these results indicate a fundamentally different manner of processing of peroxides by cytochrome P-450 than by the peroxidases. Thus, the possibility of close similarity of reactive oxygen intermediates in the two series is called into question.

For a set of 10 para-substituted toluene derivatives, three enzymatic constants were determined describing their interaction with purified rabbit liver microsomal P-450LM2. The three constants were the catalytic rate constant (Kcat) for hydroxylation, the apparent dissociation constant (Kd) for the enzyme-substrate complex, and the interaction energy (delta Gint) between the substrate-binding and spin-state equilibria. The para-substituents of the toluene substrates were: hydrogen, fluoro, bromo, chloro, iodo, nitro, methyl, cyano, isopropyl, and t-butyl. Linear free energy correlations were sought between the enzymatic constants and several physical constants of the individual substrate molecules. These correlations would be useful both for empirical prediction purposes and for insight into active site chemistry and mechanics. Catalytic rates were correlated by a linear combination of the Hansch pi hydrophobic constant and the Hammett sigma value. A deuterium isotope effect (DV) of 2.6 for d8-toluene compared to d0-toluene confirmed that hydrogen abstraction was partially rate-limiting with this series of substrates. Apparent dissociation constants were predicted by a linear combination of the molar volume and pi, while the spin-state interaction energies were best predicted by a linear combination of the Hansch pi hydrophobic constant and the reciprocal of the dielectric constant.

Three alicyclic compounds (D-camphor, adamantanone, adamantane) were found to be hydroxylated by the cytochrome P-450 isoenzymes P-450cam and P-450LM2. With P-450cam as the catalyst only one product was formed from each of the substrates: 5-exohydroxycamphor, 5-hydroxyadamantanone, and 1-adamantanol. With P-450LM2 as the catalyst, two or more isomeric products were formed from each substrate: 3-endo-, 5-exo-, and 5-endo-hydroxycamphor; 4-anti- and 5-hydroxyadamantanone; and 1- and 2- adamantanol. The products from P-450cam hydroxylations were found to be isosteric with one another, suggesting that each of them was attacked at a topologically congruent position within a rigid enzyme-substrate complex. The distribution of products from P-450LM2 hydroxylations, on the other hand, were similar to the distributions expected during solution-phase hydroxylations. Thus, it would appear that the complex which P-450LM2 forms with its substrate allows considerable movement of the substrate molecule, such that most of the hydrogens in the substrate are exposed to the enzymatic hydrogen abstractor. Under these conditions, the distribution of products more nearly reflects the rank order of chemical reactivities of the various hydroxylatable positions, with only a moderate protein-based steric constraint being expressed. These suggestions were also evident in the tightness of binding of the substrates to the two enzymes and in the magnitude of coupling between the substrate binding and the spin-state equilibria. Thus, the product from P-450cam-catalyzed hydroxylation may be predicted by a consideration of the relation of the topology of the prospective substrate to that of D-camphor. The products from P-450LM2-catalyzed hydroxylations, on the other hand, may be approximately predicted from the chemical reactivities of the various abstractable hydrogens in the prospective substrate.

We have developed a bone organ culture system that mineralizes in vitro. Fetal rat parietal bones (20 days old) were cultured in a chemically defined serum-free medium containing physiological 3 mM phosphate. During 5 days in culture, calcium content increased from 26 to 55 micrograms and dry weight increased from 137 to 194 micrograms. After 2 days in vivo, the calcium content of the parietal bone showed a comparable increase to 49 micrograms and dry weight increased to 183 micrograms. During culture, the mineralized bone area in thick sections increased from 11 to 23%, which paralleled the doubling in calcium content. Fluorescent calcein labeling during the 5 day culture period demonstrated that calcification occurs in an ordered pattern. Protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP). The percentage collagen synthesis decreased from 17.5% at 0 time to 5.0% at 2 days and then increased to 9.4% at 5 days of culture. Varying the inorganic phosphate concentration in the medium or adding beta-glycerol phosphate was found to affect mineralization. After 5 days in culture, bones treated with 1 mM phosphate exhibited a large region of unmineralized osteoid with only a 23% increase in calcium content compared with 112% in control (3 mM phosphate) bones and a 28% increase in dry weight compared with a 40% increase in control. Treatment for 5 days with 6 mM phosphate or 1, 3, or 10 mM beta-glycerol phosphate had no significant effect on dry weight compared to control bones. However, bone calcium content increased significantly from 55 +/- 5 micrograms in control cultures to 105 +/- 7 with 6 mM phosphate, 74 +/- 6 with 3 mM beta-glycerol phosphate, and 75 +/- 5 micrograms with 10 mM beta-glycerol phosphate. Calcified area measured by histomorphometry was also significantly greater than in control bones, but this was mainly due to ectopic calcification in the periosteum, representing from 23 to 74% of the total increase in calcified matrix in bones cultured with 6 mM phosphate or 1-10 mM beta-glycerol phosphate. Ultrastructural analysis demonstrated that ectopic calcification was associated with cell death and debris. Therefore, calcification with beta-glycerol phosphate and high concentrations of inorganic phosphate differed from mineralization in vivo or in bones cultured with a physiologically concentration of phosphate.

In the presence of peroxyphenylacetic acid and a hydroxylatable substrate, cytochrome P-450LM2 catalyzes two reactions which proceed concurrently, decarboxylation of the peroxyacid and hydroxylation of substrate. While the first process is definitely the result of homolytic cleavage of the peroxide O--O bond, the second may involve a different mechanistic pathway. We have undertaken to determine the relationship between these two processes through a kinetic analysis of the system. Seven different mechanistic schemes are advanced to account for the two processes. The two reactions were found to have different apparent Michaelis constants for peroxyacid and different inhibition constants for cyanide. Since the ratio of the two products is saturable at high substrate concentrations, the decarboxylation reaction can proceed from both substrate-bound and substrate-free enzyme. By appropriate manipulation of the rate equations it is possible to derive expressions for the ratio of hydrogen abstraction rate constants for a series of p-substituted toluene substrates compared to toluene. The nature of the correlation of these expressions with delta in a Hammett plot allowed some of the schemes to be eliminated. After consideration of all the data, we concluded that the processes of decarboxylation and hydroxylation occur in separate pathways and do not involve any common intermediate beyond the ferric resting state of the enzyme.