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Bridging large bone defects with mesenchymal stromal cells-seeded scaffolds remains a big challenge in orthopedic surgery, due to the lack of vascularization. Within such a cell-scaffold construct, cells are exposed to ischemic conditions. When human mesenchymal stem cells (hMSCs) encounter hypoxic conditions, they show higher cell proliferation than at ambient oxygen levels. However, when hMSCs are exposed to prolonged ischemia, cell proliferation ceases completely. Exposure of hMSCs to hypoxic conditions is known to result in the transcription of angiogenic factors (AGF), which can promote the development of new blood vessels. In this study, we investigated at which oxygen level hMSC proliferation and the transcription of AGF were optimal. Human bone marrow-derived hMSCs were cultured at 0.1, 1, 2, 3, 4, 5, and 21% oxygen. Cell proliferation over 14 days was assayed using a DNA quantification method. hMSC metabolic activity over 14 days was measured using a MTT test. Quantitative RT-PCR was used to assess mRNA levels of angiogenic factors at the tested oxygen percentages. hMSCs showed the highest cell proliferation rate at 1% oxygen. The highest corrected cell metabolic rate was found at 21% oxygen, followed by 2% oxygen. HIF1 transcription did not increase under hypoxic conditions compared to 21% oxygen conditions. However, transcription of VEGF and ANG-1 was significantly higher at 2% oxygen than at 21% O-2. The optimum oxygen range at which hMSCs proliferated rapidly and angiogenic factors ANG-1 and VEGF simultaneously came to expression was from 1 to 2% oxygen.

Background: Idiopathic epiretinal membrane (iERM) is a fibrocellular membrane that proliferates on the inner surface of the retina at the macular area. Membrane contraction is an important sight-threatening event and is due to fibrotic remodeling. Methods: Analysis of the current literature regarding the epidemiology, clinical features, and pathogenesis of iERM and fibrotic tissue contraction. Results: Epidemiologic studies report a relationship between iERM prevalence, increasing age, and posterior vitreous detachment. Clinically, iERM progresses through different stages characterized by an increased thickness and wrinkling of the membrane. Pathophysiologically, iERM formation is a fibrotic process in which myofibroblast formation and the deposition of newly formed collagens play key roles. Anomalous posterior vitreous detachment may be a key event initiating the formation of iERM. The age-related accumulation of advanced glycation end products may contribute to anomalous posterior vitreous detachment formation and may also influence the mechanical properties of the iERM. Conclusion: Remodeling of the extracellular matrix at the vitreoretinal interface by aging and fibrotic changes, plays a significant role in the pathogenesis of iERM. A better understanding of molecular mechanisms underlying this process may eventually lead to the development of effective and nonsurgical approaches to treat and prevent vitreoretinal fibrotic diseases.

Purpose: To investigate the identity of collagens and cellular components in the epiretinal membrane (ERM) associated with full-thickness idiopathic macular hole and their clinical relevance. Methods: Pars plana vitrectomy with the peeling of internal limiting membrane and ERM was performed by 2 surgeons in 40 eyes with idiopathic macular holes. The clinical data were reviewed and the surgical specimens were processed for flat-mount and immunohistochemical analysis. Results: Epiretinal membrane is a GFAP-positive gliotic and fibrotic scar which contains newly formed Type I, III, and V collagens. Type VI collagen was not observed. Colocalization studies found cells coexpressing GFAP/CRALBP, GFAP/alpha-SMA, and alpha-SMA/CRALBP, which are consistent with transdifferentiation of Muller cells into fibroblasts and myofibroblasts. The clinically significant ERMs can be divided into two groups according to the amount of cells in ERM: sparse cellular proliferation and dense cellular proliferation. The latter group is associated with a higher chance of surgical difficulty during internal limiting membrane peeling (P = 0.006). Preoperative and postoperative visual function were not affected by the density of the cellular proliferation. Conclusion: Retinal glial cells, probably transdifferentiated Muller cells, are involved in the formation of full-thickness macular hole-associated ERMs by a gliotic and fibrotic process. Such ERMs contain newly formed Type I, III, and V collagen depositions. The cell density of ERM affects its biomechanical properties and determines the difficulty of ERM peeling.

Photocatalytic-activation of anodized TiO2-surfaces has been demonstrated to yield antibacterial and tissue integrating effects, but effects on simultaneous growth of tissue cells and bacteria in co-culture have never been studied. Moreover, it is unknown how human-bone-marrow-mesenchymal-stem (hBMMS) cells, laying the groundwork for integration of titanium implants in bone, respond to photocatalytic activation of anodized TiO2-surfaces. Photocatalytically-activated, anodized titanium and titanium-alloy surfaces achieved 99.99% killing of adhering Staphylococcus epidermidis and Staphylococcus aureus, an effect that lasted for 30 days of storage in air. Surface coverage by osteoblasts was not affected by photocatalytic activation of anodized TiO2-surfaces. Co-cultures of osteoblasts with contaminating S. epidermidis however, enhanced surface coverage on photocatalytically-activated, anodized titanium-alloy surfaces. hBMMS cells grew less on photocatalytically-activated, anodized titanium surfaces, while not at all on photocatalytically-activated, anodized titanium-alloy surfaces and did not survive the presence of contaminating staphylococci. This reduced surface coverage by hBMMS cells disappeared when photocatalytically-activated, anodized titanium-alloy surfaces were exposed to buffer for 60 min, both in absence or presence of contaminating S. aureus. Consequently, it is concluded that photocatalytically-activated, anodized titanium and titanium-alloy surfaces will effectively kill peri-operatively introduced staphylococci contaminating an implant surface and constitute an effective means for antibiotic prophylaxis in cementless fixation of orthopaedic hardware.

Purpose: There is a growing interest in markers for cartilage degradation in synovial joints because of their potential diagnostic and prognostic value. Therefore, the aim of this study was to identify valuable degradation markers for temporomandibular joint (TMJ) osteoarthritis (OA) by comparing the relative concentrations of carboxyterminal telopeptides type I and II (CTX-I and II), cartilage oligomeric matrix protein (COMP), and prostaglandin E-2 (PGE(2)) in the synovial fluid (SF) of TMJs with OA with those of healthy symptom-free TMJs.Materials and Methods: In this cross-sectional case-control study, participants were recruited from the University Medical Center Groningen (Groningen, the Netherlands). Cases were defined as patients with TMJ OA, and control patients had symptom-free TMJs. The outcome variables were the relative concentrations of CTX-I, CTX-II, COMP, and PGE(2) in osteoarthritic TMJ SF compared with symptom-free joints. An independent-samples Mann-Whitney U test was used to compare the relative concentrations.Results: Thirty cases (9 male, 21 female; mean age, 40.1 yr; standard deviation, 15.3 yr) and 10 controls (5 male, 5 female; mean age, 30.3 yr; standard deviation, 10.8 yr) were studied. No significant differences in relative concentrations of CTX-I (P = .548), CTX-II (P = .842), COMP (P = .140), and PGE(2) (P = .450) were found between the groups. Unexpected low relative concentrations of CTX-I and high relative concentrations of CTX-II were observed.Conclusions: Assumed changes in the SF concentration of CTX-I, CTX-II, COMP, and PGE(2) in TMJ OA seem to occur proportionally. Furthermore, the unexpected large contribution of CTX-II suggests that this marker may be useful to quantify cartilage degradation in TMJ OA. (C) 2013 American Association of Oral and Maxillofacial Surgeons

Biopolymers are an attractive class of compounds for being used in biomedical applications as they are widely available from biomass. Their drawback is the lack of mechanical stability and the ability to tune this properly. Covalent chemical cross-linking is an often used approach but it limits usability due to legislation as well as the need of advanced and specialized knowledge by end users such as clinicians. Here, increased and tunable mechanical properties are achieved of alginate-based hydrogels with non-covalent approaches using linear polyethyleneimine (LPEI) as a polyelectrolyte rather than only multivalent metal ions (Ca2+). Gel stiffness increases with increasing LPEI content. Gel morphology changes from a thin fibrous mesh for alginate-Ca2+ to thicker fibrous networks when LPEI is introduced. The gels are able to efficiently release encapsulated small molecular dyes and the gels are able to host cells. For the cell encapsulation human skin fibroblasts (HSkF) and human bone marrow-derived mesenchymal stem cells (hBM-MSC) are used. HSkF can be successfully incorporated without diminished viability while the matrix components and gel preparation method are not compatible with hBM-MSC. The newly developed alginate-based system is regarded as a potential candidate for wound dressing materials.

Cartilage has a poor regenerative capacity. Tissue-engineering approaches using porous scaffolds seeded with chondrocytes may improve cartilage repair. The aim of this study was to examine the effect of pore size and pore interconnectivity on cartilage repair in osteochondral defects treated with different scaffolds seeded with allogenic chondrocytes. Scaffolds consisting of 55wt% poly(ethylene oxide terephthalate) and 45wt% poly(butylene terephthalate) (PEOT/PBT) with different pore sizes and interconnectivities were made, using a compression moulding (CM) and a three-dimensional fibre (3DF) deposition technique. In these scaffolds, allogenic chondrocytes were seeded, cultured for 3weeks and implanted in osteochondral defects of skeletally mature rabbits. At 3weeks no difference in cartilage repair between an empty osteochondral defect, CM or 3DF scaffolds was found. Three months post-implantation, cartilage repair was significantly improved after implantation of a 3DF scaffold compared to a CM scaffold. Although not significant, Mankin scores for osteoarthritis (OA) indicated less OA in the 3DF scaffold group compared to empty defects and CM-treated defects. It is concluded that scaffold pore size and pore interconnectivity influences osteochondral repair and a decreased pore interconnectivity seems to impair osteochondral repair. Copyright (c) 2012 John Wiley & Sons, Ltd.

PURPOSE: Articular cartilage has some capacity for self-repair. Clinically used low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments were compared in their potency to prevent degeneration using an explant model of porcine cartilage.METHODS: Explants of porcine cartilage and human osteoarthritic cartilage were cultured for four weeks and subjected to daily LIPUS or PEMF treatments. At one, two, three and four weeks follow-up explants were prepared for histological assessment or gene expression (porcine only).RESULTS: Non-treated porcine explants showed signs of atrophy of the superficial zone starting at one week. Treated explants did not. In LIPUS-treated explants cell clusters were observed. In PEMF-treated explants more hypertrophic-like changes were observed at later follow up. Newly synthesized tissue was present in treated explants. Gene expression profiles did indicate differences between the two methods. Both methods reduced expression of the aggrecan and collagen type II gene compared to the control. LIPUS treatment of human cartilage samples resulted in a reduction of degeneration according to Mankin scoring. PEMF treatment did not.CONCLUSIONS: LIPUS or PEMF prevented degenerative changes in pig knee cartilage explants. LIPUS reduced degeneration in human cartilage samples. LIPUS treatment seems to have more potency in the treatment of osteoarthritis than PEMF treatment.

Biomaterial-implants are frequently used to restore function and form of human anatomy. However, the presence of implanted biomaterials dramatically elevates infection risk. Paradoxically, dental-implants placed in a bacteria-laden milieu experience moderate failure-rates, due to infection (0.0-1.1 %), similar to the ones of joint-arthroplasties placed in a near-sterile environment (0.1-1.3 %). Transcutaneous bone-fixation pins breach the immune-barrier of the epidermis, exposing underlying sterile-tissue to an unsterile external environment. In contrast to dental-implants, also placed in a highly unsterile environment, these pins give rise to relatively high infection-associated failure-rates of up to 23.0 %. Herein, we attempt to identify causes as to why dental-implants so often succeed, where others fail. The major part of all implants considered are metal-made, with similar surface-finishes. Material choice was therefore discarded as underlying the paradox. Antimicrobial activity of saliva has also been suggested as a cause for the success of dental-implants, but was discarded because saliva is the implant-site-fluid from which viable bacteria adhere. Crevicular fluid was discarded as it is largely analogous to serum. Instead, we attribute the relative success of dental-implants to (1) ability of oral tissues to heal rapidly in the continuous presence of commensal bacteria and opportunistic pathogens, and (2) tolerance of the oral immune-system. Inability of local tissue to adhere, spread and grow in presence of bacteria and an intolerant immune-system are identified as the likely main causes explaining the susceptibility of other implants to infection-associated failure. In conclusion, it is the authors' belief that new anti-infection strategies for a wide range of biomaterial-implants may be derived from the relative success of dental-implants.

Posterior capsular opacification (PCO) is a common complication of cataract surgery. The development of PCO is due to a combination of the processes of proliferation, migration, and transdifferentiation of residual lens epithelial cells (LECs) on the lens capsule. In the past decades, various forms of PCO prevention have been examined, including adjustments of techniques and intraocular lens materials, pharmacological treatments, and prevention by interfering with biological processes in LECs. The only method so far that seems effective is the implantation of an intraocular lens with sharp edged optics to mechanically prevent PCO formation. In this review, current knowledge of the prevention of PCO will be described. We illustrate the biological pathways underlying PCO formation and the various approaches to interfere with the biological processes to prevent PCO. In this type of prevention, the use of nanotechnological advances can play a role.

ObjectivesOur study compared two novel, biodegradable poly(trimethylene carbonate) (PTMC) barrier membranes to clinically applied barrier membranes in maintaining volume of block autologous bone grafts in a rat mandible model.Material and methodsTwo hundred and forty rats were included in this study. Block autologous bone grafts of 5mm in diameter were harvested from the mandibular angles and transplanted onto the contralateral side. The bone grafts were either covered with a membrane or left uncovered. The applied membranes included pure PTMC membranes, biphasic calcium phosphate (BCP) incorporated PTMC composite membranes, expanded poly(tetrafluoroethylene) (e-PTFE) membranes (Tex) and collagen membranes (Geistlich Bio-Gide). After 2, 4 and 12weeks, the rat mandibles were retrieved and analysed by histological evaluation and CT quantification.ResultsThe histological evaluation revealed that in time the block autologous bone graft was well integrated to the recipient bone via gradually maturing newly formed bone and did not show signs of resorption, independent of membrane coverage or types of membrane. CT quantification showed the volume of the bone graft and recipient bone together was maintained by new bone formation and recipient bone resorption.ConclusionsOur study showed that the use of PTMC membranes and PTMC-BCP composite membranes resulted in similar bone remodelling to the collagen membranes and e-PTFE membranes and that the use of barrier membranes did not interfere with bone remodelling of the bone grafts and recipient bones. However, the used barrier membranes seemed not to contribute in maintaining the volume of block autologous bone grafts.

The aim of this study was to investigate whether polymeric biomaterials can be designed such that they become suitable for surgical closure of medium-sized perforations in the cornea, the transparent tissue in the front of the eye. Such a biomaterial must meet stringent requirements in terms of hydrophilicity, strength, transparency, flexibility, and biocompatibility. Four different copolymers of n-butyl methacrylate (BMA) and hexa(ethylene glycol) methacrylate (HEGMA) were prepared and characterized. Poly(BMA) was made as a reference material. Physicochemical properties were measured (contact angles, glass-transition temperatures, thermal degradation, water uptake and swelling), and cytotoxicity in vitro was assessed with a MTT test. Moreover, the interaction between the materials and cultured human corneal epithelial cells was studied. The copolymers may be useful for temporary closure of corneal perforations.

The quantity of bone formed in cylinders of a newly developed erodible copolymer, Polyactive (PA60/40) was examined. PA60/40 was implanted in three different bone locations in the rabbit: in the cortex, in bone marrow and in trabecular subchondral bone. Bony ingrowth was assessed after 4, 8, 26 and 52 w after the operation and investigated by histology and image analysis. The ingrowth of bone was observed in PA60/40 placed in the cortex from 4 w onwards. After 8 w, more than 90% of the pores of the biomaterial were filled with dense bone. In bone marrow, initially some bone formation was seen. After 26 w, all newly formed bone was resorbed. Subchondral bone formation was less than in the cortex of the femur, but somewhat comparable to the amount of bone found in healthy trabecular bone. Bone formation appeared not to be affected by the degradation of the biomaterial. It was concluded that Polyactive is a suitable bone graft substitute. Bone formation within PA60/40 is site-dependent and this follows Wolff 's law.

The biocompatible, osteoconductive and resorbable polymer Polyactive (PA) was investigated for its performance as a bone-graft substitute. The model consisted of a 4 mm borehole, 1.5 cm distal of the major trochanter in both femurs of a rabbit, of which one was filled with a cylinder of porous PA. The other was left untreated. PA70/30 and PA60/40 were investigated, both before and after being incubated with allogenic bone marrow. Analyses were performed after 4, 8, 26 and 52 weeks and comprised dual energy X-ray absorptiometry (DXA) and image analysis of histological sections. DXA revealed an increased bone mineral density in the filled defects compared to the controls, both at the defect and immediately proximal and distal of the defect. Histology showed that gap-bridging had occurred within 8 weeks, with 80%-90% of the pores of PA70/30 and PA60/40 occupied by new bone, and an intimate bone-PA contact. PA70/30 seemed to be more suitable compared to PA60/40, in that the highest amount of bone was formed within the shortest period of time. Incubation of PA with allogenic bone marrow resulted in inflammatory reactions at the sites of implantation, which delayed bone growth, but did not prevent it. It was concluded that PA70/30 and PA60/40 are suitable bone-graft substitutes.

The usefulness of thionin for staining cartilage sections embedded in glycol methacrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin O stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin O, thionin did not show metachromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.

Abstr A ct . We examined the hypothesis that the circumferential osseous apposition around a HA-coated implant forms a protective barrier against articular wear debris. Sixty-five hydroxyapatite-coated total hip arthroplasties in 57 patients (age

Rat patellae were preincubated with culture medium M199 for one hour and then with either fresh culture medium or Ringer's solution, Ringer lactate, Ringer glucose, normal saline or Betadine for another hour. The rate of proteoglycan synthesis in the articular cartilage was then measured by uptake of 35SO4 for the next 16 hours. Cartilage metabolism was inhibited by all of the solutions even after a recovery time of 16 hours. The inhibition was by 5% for Ringer's solution, 10% for Ringer glucose (p < 0.01), 20% for saline and Ringer lactate (p < 0.001) and 55% for Betadine (p < 0.001). Ringer's solution is therefore the best choice for joint irrigation during arthroscopy or other procedures.

Tissue engineering of articular cartilage in order to restore the function of degenerated, diarthrodial joints is currently widely under investigation. The results obtained thus far indicate that proper control of the differentiation of the cells used for this purpose is essential to produce and maintain a hyaline-like matrix. In this study, a procedure is described by which differentiation of chondrocytes in vitro and ex vivo can be studied. The method involves quantitative assessment of mRNA for different collagens, which are markers for differentiation of chondrocytes, by competitive PCR. In a culture system employing human osteoarthritic chondrocytes, mRNAs for the alpha(1)-chains of collagen types I, II and X are quantified. The procedure is fast, specific and sensitive. However, several controls should be included to ascertain the reliability of the assessment. (C) 1998 Kluwer Academic Publishers.

Eighty-eight patients with articular cartilage defects in the knee were treated by perichondrial arthroplasty between 1986 and 1992. An autogenous strip of costal perichondrium was fixed in place with fibrin glue, followed by immobilisation, continuous passive motion, and partial weightbearing. The results were evaluated using the Hospital for Special Surgery Score for knee function, radiographs, arthroscopy and the patient's subjective opinion. The results after a mean follow-up of 52 months were good in 38%, fair in 8% and poor in 55%. Previous drilling or shaving of a defect, concomitant osteoarthritis, older age and a long history of complaints proved to be contraindications. Good results were seen in 91% of isolated defects. Perichondrial arthroplasty can be beneficial in the repair of cartilage defects. It will reduce symptoms in carefully selected cases, and avoid more extensive operations for osteoarthritis.

A xenograft of costal rabbit perichondrium was trans-planted to an articular cartilage defect in a sheep knee. After 12 weeks, cartilage was formed with increased calcification of the basal layer and a mean of 74 percent collagen type II. the synovium did not show any infiltration, indicating the absence of any immunologic reaction.

The effect of piroxicam on the metabolism of healthy and osteoarthrotic (OA) chondrocytes was studied in vitro. The chondrocytes were obtained from five healthy, five moderately OA, and four severely OA hips or knees. The chondrocytes were cultured in a high-density, short-term in vitro model. In this culture, the healthy chondrocytes as well as the OA chondrocytes retain their metabolic properties. Piroxicam was used in concentrations ranging from 0 to 10 micrograms/ml, which is comparable to the concentrations reached in vivo after oral administration. In cultures of healthy chondrocytes, piroxicam inhibited proliferation and synthesis of proteoglycans. The metabolism of moderately damaged chondrocytes was not influenced by piroxicam. In severely damaged chondrocytes, the proliferation was significantly inhibited by piroxicam. In order to avoid the possible negative side effects of piroxicam on the metabolism of healthy and severely OA chondrocytes, piroxicam treatment of an OA joint with synovitis should be restricted to the period of the effusion.

The effects of the sulfate- and FCS concentration on the rate of synthesis and the biochemical properties of glycosaminoglycans, synthesized in bovine articular cartilage in vitro, were studied. 20% FCS in the culture medium stimulated the rate of synthesis. In media without FCS, the rate of synthesis decends from day 0 on. The differences in incorporation rates of [35S]-sodium sulfate and 1,6-[3H]-glucosamine-HCl into glycosaminoglycans in serum free media containing 9 microM and 900 microM sulfate were used to discuss the inorganic sulfate concentration in cartilage. In 9 microM sulfate medium, the newly synthesized glycosaminoglycans contain higher levels of unsulfated disaccharides than the endogenous glycosaminoglycans. In each culture medium, the ratio 6-sulfated disaccharides to 4-sulfated disaccharides of the newly synthesized glycosaminoglycans becomes higher after 3 days in culture. The glycosaminoglycan synthesis is underestimated, when chondrocytes are cultured in media containing less than 200 microM sulfate.

The effects of various proteoglycan samples, isolated from human articular cartilage of different ages, on the rate of the lateral growth phase of the fibril formation of collagen type II were studied by turbidimetry. In general, proteoglycan aggregates accelerate fibrillogenesis, whereas non-aggregating proteoglycans retard this process. The only exception were non-aggregating proteoglycans from very young cartilage, which stimulated the fibril formation strongly. The extent of stimulation by proteoglycans from hip and knee cartilage were compared. The effects of non-aggregating proteoglycans dominate those of aggregated proteoglycans. Chondroitinase ABC digestion of proteoglycan samples did not change the effects on the fibrillogenesis of collagen type II, when these samples were isolated from 18 years-old knee cartilage. The collagen fibril formation was less stimulated in the presence of ABC-ase digested proteoglycan samples from 0-3 month-old knee cartilage, suggesting a primary role for keratan sulphate and a possible influence of chondroitin sulphate when keratan sulphate is not present. Only proteoglycans from very old cartilage were able to reduce the amount of collagen fibrils formed in vitro. Proteoglycans could not be detected bound to the fibril pellet despite the fact that part of the pellet was not dissolvable in acetic acid. It is concluded that proteoglycans may play a regulatory role in collagen type II fibril formation in articular cartilage.

In response to degenerative changes (tissue disorganization) in articular cartilage, chondrocytes synthesize proteoglycans which differ in several aspects from those found in healthy cartilage [1]. The main object of this study was to investigate the effects of the severity of tissue disorganization on the nature of the newly synthezised proteoglycans. The proteoglycans synthesized by chondrocytes in explant cultures, high density (pellet) and monolayer cultures are compared. The study is focussed on the degree of sulphation and the position of sulphate in chondroitin sulphate chains ofproteoglycans synthesized in vitro.

Proteoglycans (A1 fractions) were extracted with 4M guanidine hydrochloride (GuHCl) from human articular cartilage samples of a wide age range. Distinctions were made between hip and knee, and upper and lower layers. The residues of these extractions were digested with purified collagenase, and a second extraction with 4M GuHCl was performed, which yielded appreciable amounts of proteoglycans. When pro-teoglycans from second extractions were compared with those from first extractions, the following changes were observed: an increase in chondroitin sulfate; a relative decrease in keratan sulfate; a decrease in protein content; and a decrease in the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate. The same changes were found when nonaggregating proteoglycans were compared with proteoglycan aggregates, when proteoglycans from young cartilage were compared with those from mature cartilage, when proteoglycans from knee cartilage were compared with those from hip cartilage, and when proteoglycans from upper layers of cartilage were compared with those from deeper layers. It is suggested that the differences found between first and second extractions of cartilage, between upper and lower layers of cartilage, and between knee and hip cartilage are caused by variations in the relative amount of nonaggregating proteoglycans and/or variations in proteoglycan size.

High-density cultures of chick embryonic chondrocytes were exposed to intermittent compressive force (ICF) of physiologic magnitude for 24 hours. Proteoglycan synthesis was significantly increased in chondrocyte cultures exposed to ICF as compared with control cultures. Similar effects were found in explants of epiphyseal cartilage. Proteoglycans extracted with guanidine-HCI from cultures exposed to ICF aggregated better with hyaluronic acid than did control cultures, as shown by Sepharose 2B gel chromatography. In addition, the amount of non-extractable proteoglycans was increased in ICF cultures. We conclude that ICF not only increases the synthesis of proteoglycans but also improves the aggregating capacity of proteoglycans and the coherence of proteoglycans with other matrix components. High-density cultures of epiphyseal chondrocytes provide a suitable model to study the processes involved in the perception of and the subsequent cellular response to compressive force by cartilage.