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This study aimed to investigate the innovative antigliotic guiding regenerative gel (AGRG) as reviving matrix for reconnection of spinal cord defect in rat models of complete acute and chronic spinal cord injury (SCI). In acute SCI, a 2 mm segment of the spinal cord (SC) was removed at Th7-Th8. Then AGRG was injected to the gap or left untreated. In chronic SCI, a 1 mm segment of the spinal cord (SC) was removed at Th7-Th8. One month later, the injured area was cleaned from connective and scar tissue, creating a gap of 2-3 mm. Then, AGRG was injected to the gap or left untreated. Functional, electrophysiological, histological and immunohistochemical assessments were performed. In acute SCI, at week 24, 75% of AGRG group showed a somatosensory evoked potential (SEP) signal. Appearance of myelin basic protein (MBP) was observed in the injured area in the AGRG group (p 0.1), compared to the untreated group. In chronic SCI, 24 weeks after 2

Hollow nerve guidance conduits are approved for clinical use for defect lengths of up to 3 cm. This is because also in pre-clinical evaluation they are less effective in the support of nerve regeneration over critical defect lengths. Hydrogel luminal fillers are thought to improve the regeneration outcome by providing an optimized matrix inside bioartificial nerve grafts. We evaluated here a modified hyaluronic acid-laminin-hydrogel (M-HAL) as luminal filler for two clinically approved hollow nerve guides. Collagen-based and chitosan-based nerve guides were filled with M-HAL in two different concentrations and the regeneration outcome comprehensively studied in the acute repair rat sciatic nerve 15 mm critical defect size model. Autologous nerve graft (ANG) repair served as gold-standard control. At 120 days post-surgery, all ANG rats demonstrated electrodiagnostically detectable motor recovery. Both concentrations of the hydrogel luminal filler induced improved regeneration outcome over empty nerve guides. However, neither combination with collagen- nor chitosan-based nerve guides resulted in functional recovery comparable to the ANG repair. In contrast to our previous studies, we demonstrate here that M-HAL slightly improved the overall performance of either empty nerve guide type in the critical defect size model.

Background and Aims:The aim of this study was to investigate the innovative guiding regenerative gel (GRG) and antigliotic GRG (AGRG) fillings for nerve conduits, prepared with Food and Drug Administration (FDA)-approved agents and expected to provide an alternative to autologous nerve graft and to enable reconnection of massive nerve gaps in a rabbit model of chronic peripheral nerve injury with massive loss defect that simulates the human condition of chronic injury with a large gap.Methods:The components and dosimetry for GRG and AGRG formulations were investigatedin vitroon nerve cell culture andin vivoon 10-mm reconstructed sciatic nerves of 72 rats using different concentrations of agents and completed on a rabbit model of delayed (chronic) complete peripheral nerve injury with a 25-mm gap. Forty rabbits underwent delayed (9 weeks after complete injury of the tibial portion of the sciatic nerve) nerve tube reconstruction of a gap that is 25 mm long. GRG and AGRG groups were compared with autologous and empty tube reconstructed groups. Rats and rabbits underwent electrophysiological and histochemical assessments (19 weeks for rats and 40 weeks for rabbits).Results:Application of AGRG showed a significant increase of about 78% in neurite length per cell and was shown to have the most promising effect on neuronal outgrowth, with total number of neurites increasing by 4-fold. The electrophysiological follow-up showed that AGRG treatment is most promising for the reconstruction of the tibial portion of the sciatic nerve with a critical gap of 25 mm. The beneficial effect of AGRG was found when compared with the autologous nerve graft reconstruction. Thirty-one weeks post the second surgery (delayed reconstruction), histochemical observation showed significant regeneration after using AGRG neurogel, compared with the empty tube, and succeeded in significantly regenerating the nerve, as well as the autologous nerve graft, which was almost similar to a healthy nerve.Conclusion:We demonstrate that in the model of delayed peripheral nerve repair with massive loss defect, the application of AGRG led to a stronger nerve recovery and can be an alternative to autologous nerve graft.

In the current study we investigated the suitability of a novel hyaluronic acid–laminin hydrogel (HAL) as luminal filler and carrier system for co-transplanted cells within a composite chitosan-based nerve graft (CNG) in a rat critical nerve defect model. The HAL was meant to improve the performance of our artificial nerve guides by giving additional structural and molecular support to regrowing axons. We filled hollow CNGs or two-chambered nerve guides with an inserted longitudinal chitosan film (CNG[F]s), with cell-free HAL or cell-free HA or additionally suspended either naïve Schwann cells (SCs) or fibroblast growth factor 2-overexpressing Schwann cells (FGF2-SCs) within the gels. We subjected female Lewis rats to immediate 15 mm sciatic nerve gap reconstruction and comprehensively compared axonal and functional regeneration parameters with the gold standard autologous nerve graft (ANG) repair. Motor recovery was surveyed by means of electrodiagnostic measurements at 60, 90, and 120 days post-reconstruction. Upon explantation after 120 days, lower limb target muscles were harvested for calculation of muscle-weight ratios. Semi-thin cross-sections of nerve segments distal to the grafts were evaluated histomorphometrically. After 120 days of recovery, only ANG treatment led to full motor recovery. Surprisingly, regeneration outcomes revealed no regeneration-supportive effect of HAL alone and even an impairment of peripheral nerve regeneration when combined with SCs and FGF2-SCs. Furthermore, complementary in vitro studies, conducted to elucidate the reason for this unexpected negative result, revealed that SCs and FGF2-SCs suspended within the hydrogel relatively downregulated gene expression of regeneration-supporting neurotrophic factors. In conclusion, cell-free HAL in its current formulation did not qualify for optimizing regeneration outcome through CNG[F]s. In addition, we demonstrate that our HAL, when used as a carrier system for co-transplanted SCs, changed their gene expression profile and deteriorated the pro-regenerative milieu within the nerve guides.

Hyaluronic acid (HA), a major component of the extracellular matrix, is an attractive material for various medical applications. Yet, its low mechanical rigidity and fast in vivo degradation hinder its utilization. Here, we demonstrate the reinforcement of HA by its integration with a low-molecular-weight peptide hydrogelator to produce a composite hydrogel. The formulation of HA with the fluorenylmethoxycarbonyl diphenylalanine (FmocFF) peptide, one of the most studied self-assembling hydrogel-forming building blocks, showing notable mechanical properties, resulted in the formation of stable, homogeneous hydrogels. Electron microscopy analysis demonstrated a uniform distribution of the two matrices in the composite forms. The composite hydrogels showed improved mechanical properties and stability to enzymatic degradation while maintaining their biocompatibility. Moreover, the storage modulus of the FmocFF/HA composite hydrogels reached up to 25 kPa. The composite hydrogels allowed sustained release of curcumin, a hydrophobic polyphenol showing antioxidant, anti-inflammatory, and antitumor activities. Importantly, the rate of curcumin release was modulated as a function of the concentration of the FmocFF peptide within the hydrogel matrix. This work provides a new approach for conferring mechanical rigidity and stability to HA without the need of cross-linking, thus potentially facilitating its utilization in different clinical applications, such as sustained drug release.

Peripheral neuropathy is one of the main complications of diabetes mellitus. The current study demonstrated the bimodal pattern of diabetic peripheral neuropathy found in the behavioral study of pain perception in parallel to the histopathological findings in dorsal root ganglia (DRGs) neurons and satellite Schwann cell basement membranes. A gradual decrease in heparan sulfate content, with a reciprocal increase in deposited laminin in the basement membranes of dorsal root ganglia Schwann cells, was shown in streptozotocin-treated rats. In addition, the characteristic biphasic pain profiles were demonstrated in diabetic rats, as shown by hypersensitivity at the third week and hyposensitivity at the tenth week post-streptozotocin injection, accompanied by a continuous decrease in the sciatic nerve conduction velocity. It appears that these basal membrane abnormalities in content of heparan sulfate and laminin, noticed in diabetic rats, may underline the primary damage in dorsal ganglion sensory neurons, simultaneously with the bimodal painful profile in diabetic peripheral neuropathy, simulating the scenario of filtration rate in diabetic kidney.

Background Aberrant glomerular polyanionic charge of glycosaminoglycans (GAGs) and sialic acid expression has been observed in proteinuric human and experimental glomerular diseases. Angiotensin-converting enzyme inhibitors (ACEI) lower proteinuria and amend renal function deterioration via hemodynamic mechanisms. We tested the hypothesis that ACEI modulate proteinuria additionally by modifying glomerular GAGs. Methods In this study, we explored the effects of the ACEI enalapril on proteinuria and GAG synthesis in puromycin aminonucleoside (PAN)-treated rats. We employed cationic colloidal gold (CCG) localization in glomerular basement membranes (GBM) to identify GAGs by electron microscopy and determined sialic acid residues by immunohistochemical staining with lectins. To clarify ACEI effects on GAG production in vitro , we studied de novo GAG synthesis into newly synthesized proteoglycans in podocytes and mesangial cells using 35 S incorporation. Cells were incubated with or without PAN, and with increasing doses of the ACEI enalaprilat. Results PAN rats developed severe proteinuria that was significantly improved by enalapril treatment. In non-treated PAN rats GBM GAGs were reduced, whereas in the enalapril-treated group GBM GAGs were significantly increased to control levels. Enalapril did not affect glomerular sialic acid. Furthermore, in cultured podocytes and mesangial cells PAN decreased de novo GAG synthesis, an effect which was significantly ameliorated by enalaprilat treatment. Conclusion Treatment with ACEI improves permselectivity properties of the glomerular capillary wall by maintaining its GAG content. This finding provides an additional new mechanism, whereby ACEI exert anti-proteinuric effects.

The microstructure and chemical composition of the calcite shell of the sea barnacle Tetraclita rufotincta (Pilsbry, 1916) were investigated using microscopic and analytical methods. The barnacle shell was separated mechanically into its three substructural units: outer, interior, and inner layers. The organic matrices of these structural parts were further separated into soluble and insoluble constituents and their characteristic functional groups were studied by FTIR. Investigation of the mechanical properties of the interior mass of the shell reveals remarkable viscoelastic behavior. In general, the mechanical behavior of the shell is a function of its geometry as well as of the material, of which it is constructed. In the case of T. rufotincta, as calcite is a brittle material, the elastic behavior of the shell is apparently related to its micro- and macroarchitecture. The latter enables the shell to fulfill its primary function which is to protect the organism from a hostile environment and enables its survival. Our detailed identification of the similarities and differences between the various structural components of the shell in regard to the composition and properties of the organic component will hopefully throw light on the role of organic matrices in biomineralization processes.

105 ISSN 1479-6708 10.2217/FNL.14.2 © 2014 Future Medicine Ltd Future Neurol. (2014) 9(2), 105–107 Traumatic injury of the PNS represents a major cause of morbidity and disability, which subsequently causes a substantial economic and social burden in a global perspective. In previous studies, peripheral nerve injuries have been estimated to affect 2.8% of all trauma patients, many of whom acquire life-long disability, resulting in an incidence of between 13 and 23 per 100,000 individuals a year (in developed countries) [1]. Although axons in the PNS harbor the potential of axonal regeneration, recovery of severe peripheral nerve injury is often unsatisfactory. This undesirable outcome is believed to be related to the process of axonal attrition and misdirection following nerve injury with anatomical disruption of the nerve tissue [2]. In recent years, many research advancements and understanding of the process involved in peripheral nerve regeneration allowed for the development of novel therapies that significantly improve patient’s outcome. The treatment of traumatic transection of peripheral nerves primarily consists of surgical intervention and anastomosis, either in the acute phase or during the chronic phase [3,4] . The gold standard autograft repair of the damaged peripheral nerve is far from being optimal and often disappointing [5]. An alternative to the use of grafts is the interposition of an artificial nerve scaffold [6–8]. Most repair scaffolds consist of a hollow tube made of polymeric materials (e.g., silicone), biologic materials (e.g., collagen), chitosan or biodegradable polymers [5–10]. The use of nerve guidance channels, sutured in between the proximal and distal nerve stumps, has been actively pursued in order to obtain better regenerative results and obviate the need for the additional surgical intervention. Since fewer epineural sutures are required for entubulation repair (since the nerve stumps are placed into the ends of the tube as opposed to simply abutting against the autograft), it is expected to cause less surgical trauma and diminish the potential of neuroma formation due to fibrosis. Moreover, guidance channels (or tubes) assist in directing axons from the proximal to the distal stump without interference from imperfectly aligned degenerating fascicles of the nerve graft or

Human body tissues, including bones, have the ability to repair, remodel, and even regenerate after injury, illness, or wear and tear. Important participants in this process are the mesenchymal stem cells (MSCs) that can undergo proliferation and differentiation to different lineages including osteoblasts. The ability to influence this process can be used to control the rate and direct the bony healing process. In this study, we examine a natural powder made of quality elk velvet antlers (QEVA) for its bioactivity effect over MSCs. Human bone marrow was taken and purified until MSCs were retrieved. These cells were grown in cultures and served as responders to examine the QEVA agent. QEVA extract in different concentrations was added along with defined cell culture medium (DCCM) and compared to fetal calf serum (FCS) or DCCM alone. An XTT Reagent kit served to evaluate cell viability, alkaline phosphatase (AP) staining to asses cell differentiation to osteoblasts and immunohistochemical staining for specific markers. Adding QEVA extract was shown to have an encouraging effect on cell growth rate compared to DCCM alone or DCCM together with 1% FCS. Combined with serum and DCCM, it also had a superior effect over serum and DCCM alone. AP staining showed QEVA extract addition to encourage differentiation to the osteogenic lineage; however, it was not superior to serum supplement. Immunohistochemical dyes were positive for osteopontin, signifying mature osteoblasts. QEVA powder extract was shown to induce propagation and osteogenic differentiation in human MSC cultures. Further studies are needed to pinpoint the exact agent responsible for these bioactivities.

Background and Objectives: The present study focuses on the effect of 780 nm laser irradiation on the growth of embryonic rat brain cultures embedded in NVR-Gel (crosslinked hyaluronic acid with adhesive molecule laminin and several growth factors). Dissociated neuronal cells were first grown in suspension attached to cylindrical microcarriers (MCs). The formed floating cell-MC aggregates were subsequently transferred into stationary cultures in gel and then laser treated. The response of neuronal growth following laser irradiation was investigated. Materials and Methods: Whole brains were dissected from 16 days Sprague–Dawley rat embryos. Cells were mechanically dissociated, using narrow pipettes, and seeded on positively charged cylindrical MCs. After 4– 14 days in suspension, the formed floating cell-MC aggregates were seeded as stationary cultures in NVRGel. Single cell-MC aggregates were either irradiated with near-infrared 780 nm laser beam for 1, 4, or 7 minutes, or cultured without irradiation. Laser powers were 10, 30, 50, 110, 160, 200, and 250 mW. Results: 780 nm laser irradiation accelerated fiber sprouting and neuronal cell migration from the aggregates. Furthermore, unlike control cultures, the irradiated cultures (mainly after 1 minute irradiation of 50 mW) were already established after a short time of cultivation. They contained a much higher number of large size neurons (P

The different clinical entities of osteochondromas, hereditary multiple exostoses (HME) and non-familial solitary exostosis, are known to express localized exostoses in their joint metaphyseal cartilage. In the current study biopsies of osteochondromas patients were screened with respect to a number of cellular and molecular parameters. Specifically, cartilaginous biopsy samples of nine HME patients, 10 solitary exostosis patients and 10 articular cartilages of control subjects were collected and cell cultures were established. Results obtained showed that one of the two HME samples that underwent DNA sequencing analysis (HME-1) had a novel mutation for an early stop codon, which led to an aberrant protein, migrating at a lower molecular weight position. The EXT-1 mRNA and protein levels in chondrocyte cultures derived from all nine HME patients were elevated, compared with solitary exostosis patients or control subjects. Furthermore, cell cultures of HME patients had significantly decreased pericellular heparan sulphate (HS) in comparison with cultures of solitary exostosis patients or control subjects. Immunohistochemical staining of tissue sections and Western blotting of cell cultures derived from HME patients revealed higher levels of heparanase compared with solitary exostosis patients and of control subjects. Further investigations are needed to determine whether the low pericellular HS levels in HME patients stem from decreased biosynthesis of HS, increased degradation or a combination of both. In conclusion, it appears that due to a mutated glycosyltransferase, the low content of pericellular HS in HME patients leads to the anatomical deformations with exostoses formation. Hence, elevation of HS content in the pericellular regions should be a potential molecular target for correction.

The increasing number of newborns requiring intubation and artificial ventilation in the sophisticated premature and intensive care units of recent years has been followed by a concomitant increase in the number of children who develop tracheal stenosis as a sequela of prolonged intubation, with a consequent increasing need for tracheal surgical repair. This study was designed to evaluate the ability of a new tissue-engineered biodegradable membrane to tightly seal significant tracheal defects.A surgically induced tracheal defect of 10 x 5 mm was repaired in rabbits using the NVR-7 membrane--a cross-linked copolymer derived from a dextran sulphate gelatin construct. The unique features of this new membrane are biocompatibility, biodegradability, elasticity, and suturability, as well as a smooth sterilization process. The animals were sacrificed and the tracheas examined at 2, 3, 4, and 8 weeks postsurgery.Seven (7) of 8 rabbits undergoing tracheal surgery survived, with a tight air seal and an almost normal airway. Macroscopic and microscopic studies of the removed specimens showed variable degrees of immunogenic reaction toward the membrane. In the long term (2-3 months), a complete regeneration of all the tracheal layers occurred, simulating the original structure and orderly arrangement of a normal trachea.The surgical correction using the above membrane enabled the operated animals to overcome any respiratory distress, adequately correcting the induced tracheal defect. From this experimental study, we conclude that the new NVR-7 membrane appears to be a promising therapeutic adjunct in the treatment of patients with tracheal defects.

One method to exogenously enhance the repair of articular cartilage is the local application of growth factors. This method is based on the chondrogenic effects of some agents and their potential ability to enhance cell migration. Human fetal chondrocytes were isolated and cultured. Their proliferation under the influence of different agents was microscopically evaluated. Fetal calf serum at 5% and 10% concentrations induced microscopically visible cell proliferation. Transforming growth factor beta one (5 and 10 ng/ml), insulin-like growth factor 1 (5 and 10 nmol/l) and leptin (1 and 2 ng/ml) accelerated proliferation of the cells towards the increasing gradient of the agents. Fibroblast growth factor beta (5 and 10 ng/ml), bone morphogenic protein two (10 ng/ml) and laminin (1 mg/ml) did not affect cell proliferation. This study suggests that different agents can play a role in the proliferation of fetal chondrocytes.

A feedback temperature-controlled laser soldering system (TCLS) was used for bonding skin incisions on the backs of pigs. The study was aimed: 1) to characterize the optimal soldering parameters, and 2) to compare the immediate and long-term wound healing outcomes with other wound closure modalities.A TCLS was used to bond the approximated wound margins of skin incisions on porcine backs. The reparative outcomes were evaluated macroscopically, microscopically, and immunohistochemically.The optimal soldering temperature was found to be 65 degrees C and the operating time was significantly shorter than with suturing. The immediate tight sealing of the wound by the TCLS contributed to rapid, high quality wound healing in comparison to Dermabond or Histoacryl cyanoacrylate glues or standard suturing.TCLS of incisions in porcine skin has numerous advantages, including rapid procedure and high quality reparative outcomes, over the common standard wound closure procedures. Further studies with a variety of skin lesions are needed before advocating this technique for clinical use.

Background: The purpose of this study is to illustrate the routes of migration of precartilaginous cells from the perichondrial ring of LaCroix, as a potential reservoir for growth-plate germ cells. Methods: Chondrocytes derived from the ring of LaCroix of young chicks’ proximal tibia were cultured in vitro and transfected with adenovirus vector containing the gene encoding for Escherichia coli (beta)-galactosidase (lacZ) gene, which allows assessment of the migratory routes of these cells. The lacZ- transfected cells were injected back into the perichondrial ring of LaCroix of young chicks’ proximal tibias. Four weeks later the migration root was assessed microscopically. Results: Injection of cells derived from the ring of LaCroix of neonate chicks, transfected in culture with adenoviruses containing LacZ reporter gene, allows the assessment of migratory potential of these cells. Stained cells were found at the outer layer of the epiphysis, particularly in areas adjacent to the perichondrial ring. Further longitudinal histopathological studies along the bone axis demonstrated a condensed layer of the stained cells arranged horizontally along parts of the physis. Conclusion: The perichondrial ring of LaCroix represents a potential reservoir of growth-plate germ cells in young chicks.

The first in vitro study testing the effect of a natural mixture extract of quality elk velvet antler (QEVA) on the development of bone marrow-derived mesenchymal stem cells (MSCs) is reported. To examine cellular responses, MSCs were seeded on gold-coated glass surfaces and grown in a medium supplemented with QEVA extract or with the osteogenic agent dexamethasone (Dex), or in unsupplemented medium as control. The MSCs were analyzed by light microscopy and confocal Raman microspectroscopy at different time intervals in the culture. The microscopy revealed that the proliferation, up to day 4, of cells treated with QEVA is higher than that of cells treated with Dex or of the control group. Raman spectroscopy revealed deposition of hydroxyapatite (HA) mineral by cells exposed to QEVA and HA precursors in cells treated with Dex, but no mineralization in the control group. The extent of mineralization for MSCs treated with QEVA increased systematically with time, up to day 14. These results indicate that QEVA enhances proliferation and promotes differentiation toward osteogenic fate more effectively than Dex, suggesting that addition of QEVA to culture media might be advantageous to bone-tissue-engineering implications. Copyright © 2005 John Wiley & Sons, Ltd.

In this work we evaluated the efficacy of biodegradable composite co-polymer guiding neurotube, based on tissue-engineering technology, for the treatment of complete peripheral nerve injury where the nerve defect is significant. The right sciatic nerve of 12 three-month-old rats was completely transected and peripheral nerve segment was removed. A 2.2-cm biodegradable co-polymer neurotube containing viscous gel (NVR-N-Gel) with survival factors, neuroprotective agents and Schwann cells was placed between the proximal and the distal parts of the transected nerve for reconnection a 2-cm nerve defect. The proximal and distal parts of the nerve were fixed into the neurotube using 10-0 sutures. Ultrasound observation showed growth of the axons into the composite neurotube 2 months after the surgery. Electrophysiological study indicated compound muscle action potentials in nine out of 12 rats, 2-4 months after peripheral nerve reconstructive surgery. The postoperative follow-up (up to 4 months) on the operated rats that underwent peripheral nerve reconstruction using composite co-polymer neurotube, showed beginning of re-establishment of active foot movements. The tube was dissolved and nerve showed complete reconnection. Histological observation of the nerve showed growth of myelinated axons into the site where a 2-cm nerve defect replaced by composite co-polymer neurotube and into the distal part of the nerve.(1) an innovative composite neurotube for reconstruction of significant loss of peripheral nerve segment is described; (2) a viscous gel, containing survival factors, neuroprotective agents and Schwann cells served as a regenerative environment for repair. Further investigations of this reconstructive procedure are being conducted.

Microarray gene expression analysis was utilized to identify genes upregulated in primary rat calvaria cultures in response to mechanical force. One of the identified genes designated CMF608 appeared to be novel. The corresponding full-length cDNA was cloned and characterized in more details. It encodes a putative 2597 amino acid protein containing N-terminal signal peptide, six leucine-rich repeats (LRRs), and 12 immunoglobulin-like repeats, 10 of which are clustered within the C-terminus. Expression of CMF608 is bone-specific and the main type of CMF608-positive cells is mesenchymal osteochondroprogenitors with fibroblast-like morphology. These cells reside in the perichondral fibrous ring of La Croix, periosteum, endosteum of normal bone as well as in the activated periosteum and early fibrous callus generated postfracture. Expression of CMF608 is notably absent from the regions of endochondral ossification. Mature bone cell types do not produce CMF608 with the exception of chondrocytes of the tangential layer of the articular cartilage, which are thought to be under constant mechanical loading. Ectopic expression of CMF608 in HEK293T cells shows that the protein is subjected to post-translational processing and its N-terminal approximately 90 kDa polypeptide can be found in the conditioned medium. Ectopic expression of either the full-length cDNA of CMF608 or of its N-terminal region in CMF608-negative ROS17/2.8 rat osteosarcoma cells results in transfected clones displaying increased proliferation rate and the characteristics of less-differentiated osteoblasts compared to the control cells. Our data indicate that CMF608 is a unique marker of early osteochondroprogenitor cells. We propose that it could be functionally involved in maintenance of the osteochondroprogenitor cells pool and its down-regulation precedes terminal differentiation.

Background and Objectives The basic characteristic property of wound closure is the immediate and long-term tensile strength (LTS). The objective of the current study was to compare tissue laser soldering to other available methods (i.e., cyanoacrylate glues and sutures) in the performance and outcome of wound closure and reparative healing process, with an emphasis on the immediate and LTS. Study Design/Materials and Methods The animals were divided into three groups according to the type and details of the closure procedure. Group A: laser treatments at different temperatures were compared to sutured incisions, emphasizing the LTS after 10 days. Group B: laser soldering at 65 ± 5°C was compared to chemical glues (i.e., Histoacryl® and Dermabond®), emphasizing the immediate tensile strength (ITS). Group C: LTS of laser soldered incisions was compared to that of sutured incisions at various time intervals emphasizing LTS (3, 7, 14, 28 days). Results Group A: LTS at 60°C exhibited the highest values (0.48 MPa). Group B: no ITS difference was detected between laser soldering and chemical glues. Group C: soldered incisions at 65°C exhibited higher LTS (1.81 MPa) than that of sutured incisions (1.08 MPa) (P

Objective: To evaluate whether satellite cells injected into infarct areas in rabbits remain viable during 6 weeks follow-up and can improve cardiac function as assessed by echocardiography. Methods: Myocardial infarction was induced in 16 New Zealand white rabbits, by ligation of the marginalis sinistra artery. Tissue from gluteus muscle biopsies was dissected into small pieces and cultured. Within 2–3 weeks the cells were expanded by 2–3 orders of magnitude and were fluorescent labeled. Single cell pellets for resuspension at >106/1 ml were directly injected into the infarct areas in 8 rabbits. In 8 additional rabbits, 1 ml saline was injected (control). Regional left ventricular function was assessed weekly by 2-D echocardiography until animals were sacrificed. Analysis was performed blind and independently by two experienced echocardiographers, based on the American Society of Echocardiography scheme. Results and discussion: Six treated and five control rabbits completed the study. One week after the artery occlusion, left ventricular function scoring did not differ between groups, mean 8.7±1.6 vs 8.3±1.9 (P=0.74). At 6 weeks post-injection, echocardiographic score was significantly better in the treated group, mean 2.6±0.9 vs 6.9±2.1 (P=0.002). The treated group showed significant gradual segmental improvement between the first week up to week 6. After sacrifice, macro and microscopic transmural areas showed typical changes of myocardial infarction. Histochemical staining identified viable grafted cells in high density 6 weeks post-transplantation in all grafted hearts. Conclusion: Autologous satellite cells (skeletal myofiber), can be successfully grafted into rabbit hearts following myocardial infarction and may induce improved regional left ventricular function.

Hereditary osteochondromas are often caused by mutation in the EXT1 gene. The lesions are typified by formation of a "pseudo" growth plate like lesion growing at 60 degrees to the normal growth direction of the bone. Such lesions can be mimicked surgically by reverting the position--the polarity of the zone of LaCroix. The current study attempts to compare the pathology between EXT1 gene expression in humans and surgically created osteochondroma pathology in a rat model. Tissues of human bunion, human embryonal tissue, and human adult cartilage as well as normal rat epiphyses served as controls. Rats were operated on and a 60 degree span of the ring of LaCroix was inverted as described by Delgado (Delgado, E., Rodriguez, J. I., Serada, A., Tellez, M., and Pariagoa, R.. Clin. Orthop. 201, 251-258 (1985)). The surgically created osteochondromas were assessed by histology, histochemistry, and immunohistochemistry. The findings show that the surgically created lesions contain only a small amount of FGF receptor 3 (FGFR3) expressed on mesenchymal stem cells located in the perichondrium, as compared to the cell population carrying FGFR3 in the contralateral limb. Indian hedgehog and Bcl2 are downregulated, while BMP-2 is overexpressed in the operated limb, compared to the LaCroix ring of the contralaetral limb. The shortage, as well as the disturbed migration routes of the residual mesenchymal stem cells in surgically created osteochondromas leads eventually to resorption of the pathological elements. In search of additional markers characterizing such pathological structures composed of mesenchymal stem cells and cartilaginous and bony cells, EXT1 gene was found to be expressed in the surgically created osteochondromas, like in normal growth plates. Nitric oxide synthase was also expressed like in adult cartilage, though tumor necrosis factor alpha typifying Bunion formation was absent. In summary, surgically created osteochondromas lack the massive and continuous population of mesenchymal stem cells with Bcl2 expression. However, the small residual mesenchymal cell population gives rise to short-lived EXT1-expressing cells that disappear eventually due to spontaneous resorption.

Reconstruction of articular cartilage defects using adult osteochondral allografts is an established clinical procedure, whose principal drawback is lack of lateral integration of the grafts to the surrounding tissue. Autologous chondrocytes transplantation is a sophisticated technique requiring cell culture and a staged operation. Its main draw back is the lack of mechanical strength early on. This study was conducted in order to evaluate the possibility of using embryonal epiphyses as a cartilage reconstruction tissue. A xenogeneic human to rabbit sub-acute osteochondral defect model was designed to evaluate the possibility of allogeneic implantation in humans. The following procedures were perfomed (n = 5): transplantation of 1. live epiphyses 2. live epiphyses with autogeneic periosteum 3. de-vitalized epiphyses and 4. devitalized epiphyses with autogeneic articular chondrocytes. A fifth control group did not receive any implant. Animals in groups 1 and 2 had a viable reconstruction of the articular surface with little evidence of rejection and without pannus formation. Animals in groups 3 and 4 became severely arthritic and the graft was resorbed. Nitric oxide synthase accumulation was reduced in group 1 and 2 as compared to groups 3, 4, and 5, indicating a joint preserving function of the epiphyseal grafts. Epiphyseal grafts appear to be a feasible procedure for reconstruction of articular cartilage defects even in a xenogeneic model.

Objectives: Giant cell tumor of bone is typified by massive infiltration of a bland neoplastic stroma by osteoclasts and monocyte progenitors. The current study aimed at evaluating the nature of the neoplastic cells and the mechanisms underlying the massive giant cell recruitment. Methods: Five different giant cell tumors were evaluated by immunohistochemistry, and explant cell cultures were established from the same tumors. Antigen expression profiles of both the tumors and the derived cultures were assessed. In order to determine if the mesenchymal cells are capable of differentiating into mature osteoblasts, retinoic acid was added to cell cultures and osteocalcin and alkaline phosphatase levels were measured. The proliferative effects of the mesenchymal cells on histiocyte-like cells were evaluated using the U-937 cell line. Results: A large stromal subpopulation expresses fibroblast growth factor receptor 3 (FGF-R3), indicating a mesenchymal origin of these cells. Few cells express bone- or cartilage-specific markers. Cell cultures are predominated by mesenchymal cells, as indicated by a strong staining by FGF R3. Retinoic acid induces osteoblastic differentiation, i.e. osteocalcin expression and alkaline phosphatase production. Conditioned medium of giant-cell-tumor-derived stromal cell cultures induces proliferation of U-937 cells, derived from histiocytic lymphoma. Papain digestion and dialysis of the conditioned media indicates the effector molecule to be a protein over 40 kD in size. The giant cell tumors as well as stromal cell cultures derived from giant cell tumors express osteoprotegerin ligand, the osteoclast activator. Conclusions: The neoplastic stromal spindle-shaped subpopulation of cells in giant cell tumors are mesenchymal stem cells capable of inducing histiocyte proliferation. Retinoid acid is capable of inducing differentiation of the cells into mature osteoblasts. This should be further investigated in an in vivo model to ascertain whether induction of differentiation will prevent bone loss and retard tumor progression.

Objective. Guiding Regeneration Gel (GRG) was developed in response to the clinical need of improving treatment for peripheral nerve injuries and helping patients regenerate massive regional losses in peripheral nerves. The efficacy of GRG based on tissue engineering technology for the treatment of complete peripheral nerve injury with significant loss defect was investigated.Background. Many severe peripheral nerve injuries can only be treated through surgical reconstructive procedures. Such procedures are challenging, since functional recovery is slow and can be unsatisfactory. One of the most promising solutions already in clinical practice is synthetic nerve conduits connecting the ends of damaged nerve supporting nerve regeneration. However, this solution still does not enable recovery of massive nerve loss defect.The proposed technologyis a biocompatible and biodegradable gel enhancing axonal growth and nerve regeneration. It is composed of a complex of substances comprising transparent, highly viscous gel resembling the extracellular matrix that is almost impermeable to liquids and gasses, flexible, elastic, malleable, and adaptable to various shapes and formats.Preclinical studyon rat model of peripheral nerve injury showed that GRG enhanced nerve regeneration when placed in nerve conduits, enabling recovery of massive nerve loss, previously unbridgeable, and enabled nerve regeneration at least as good as with autologous nerve graft “gold standard” treatment.

Primary synovial chondromatosis (PSC) is a rare disorder of the synovium typified by cartilaginous nodule formation within the synovial membrane. Fibroblast growth factor receptor 3 (FGFR3) is a recently described specific marker of mesenchymal precartilaginous stem cells. Expression patterns of FGFR3 and its specific ligand, fibroblast growth factor 9 (FGF 9), were evaluated both in situ and in cell cultures. Histologically, cells at the periphery of the cartilage nodules express FGFR3 and PCNA (proliferating cell nuclear antigen). Elevated levels of FGF 9, its specific ligand, have been found in synovial fluids of patients with synovial chondromatosis. Synoviocytes but not chondrocytes from affected patients express FGF9 in culture. This pattern is absent in normal synovium and cartilage. Downregulation of FGF9 may provide a possible nonoperative therapy for PSC.

Articular cartilage lacks self-repair capacity. Currently, two methods employing autologous cells are used to stimulate repair of articular cartilage. Micro-fracture induced repair induces autologous mesenchymal cell migration from bone marrow. Autologous chondrocytes' transplantation involves in vitro expansion of chondrocytes, and later implantation. In 15 patients de-differentiated chondrocytes obtained by cartilage biopsy were compared to cells derived from repair tissue induced by micro-fracture. These patients all underwent micro-fracture during the cartilage biopsy procedure. Autologous chondrocytes' transplantation was performed at least two months later then the biopsy. Tissue bits from articular cartilage and micro-fracture repair tissue were incubated in-vitro and explant cell cultures established. The cell cultures were assessed by immunohistochemistry and induced to differentiate. Differentiation into bone tissue was stimulated by addition of basic fibroblast growth factor, ascorbate and dexamethasone. High density (micro-mass) culture was used to stimulate chondrogenesis. Both cell cultures consist of mesenchymal progenitors as indicated by fibroblast growth factor receptor 3 expression and anti-CD-34+ antibodies. However, the micro-fracture generated repair tissue consists of osteocalcin-expressing cells destined to become bone. Collagen type II expression does not occur in these cells compared to autologous chondrocytes. Inducible nitric oxide synthase expression by microfracture cells is likely to damage surrounding articular cartilage in vivo. In conclusion, cells recruited by micro-fracture are inferior for cartilage regeneration purposes to those from cartilage biopsies.

Objectives : Cartilage transplantation as a means to replace damaged articular surfaces is of interest. A major obstacle is the long-term preservation of cartilage grafts. The commonly used technique of freezing the grafts inevitably leads to cellular death. The current study compares the technique to an innovative approach using a pulsed-pressure perfusion system termed a joint simulating device (JSD), intended to simulate intra-articular mechanical forces. Methods : Human articular cartilage explants were harvested from both embryonic epiphyseal tissue and femoral heads of elderly women (over 70 years of age) undergoing a partial joint replacement (hemi-arthroplasty) and were divided in two groups: half of the samples were incubated in the JSD while the remaining half were grown in static culture within tissue culture plates. After 10 days all samples were evaluated for: (a) cell vitality as assessed by image analysis and XTT assay; (b) biosynthetic activity as expressed by radioactive sulfate incorporation into glycosaminoglycans (GAG’s); and (c) proteoglycan content as assessed by alcian blue staining intensity. Results : A 10-fold increase in sulfate incorporation in samples held in the JSD compared to the static culture group was observed in embryonic cartilage. In adult cartilage culture in the JSD elevated sulfate incorporation by threefold as compared to static culture. Central necrosis was observed in specimens grown in the static culture plates, while it did not occur in the samples held in the JSD. Cell vitality as assessed by XTT assay was significantly better in the JSD group as compared to static culture. The difference was more pronounced in the embryonic specimens as compared to adult cartilage. The specimens cultured within the JSD retained proteoglycans significantly better than those cultured in static culture. Conclusions : Maintenance of cartilage specimens in a JSD was highly effective in keeping the vitality of cartilage explants in vitro over a 10-day period. A possible future application may be a long-term preservation of chondral grafts, without freezing. Aviodance of freezing of cartilage grafts, might prevent the cartilage degeneration often observed in frozen osteochondral grafts.

Achondroplasia, the most common genetic form of human dwarfism, results from a point mutation (G380R) in the gene for fibroblast growth factor receptor 3 (FGFR-3). Heterozygotes for the mutation share disproportionate, proximal shortening of the limbs, mid-face hypoplasia and relative macrocephaly due to a failure in endochondral ossification. Here we have generated transgenic mice expressing the human mutant FGFR-3 under the transcriptional control of the mouse gene. Mice that are hemizygous for the mutant human gene display disproportionate dwarfism with skeletal phenotypes remarkably similar to those of human achondroplasia. Mice that are homozygous for the transgene suffer from a profound delay in skeletal development and die at birth, similar in that respect to humans homozygous for the achondroplasia mutant gene. Microscopic analysis of long bones demonstrates growth plate morphology compatible with that of human achondroplasia cases, sharing endochondral growth inhibition with restrained chondrocyte proliferation and maturation, penetration of ossification tufts and aberrant vascularization.

Nitric oxide (NO) appears to be a final common inflammation mediator of cartilage degradation. Halting the pathological formation of excessive NO, by suppressing the inducible NO synthase (iNOS) activity, may help to preserve cartilage integrity. We used fresh ex-vivo human articular cartilage explants from normal and arthrotic joints for assessment of NO levels, as determined by its nitrite degradation products and nitric oxide synthase expression. We measured matrix proteoglycan content, assessed by image analysis of alcian blue staining, and proteoglycan synthesis, assessed by sulfate incorporation into proteoglycans. The effect of methylene blue, a nitric oxide synthase inhibitor, on matrix preservation was evaluated. Cartilage discs in vitro, derived from normal appearing joints, secreted about one tenth as much NO compared to discs derived from arthrotic cartilage. Cartilage explants showed a time-dependent reduction in the amount of aggrecan within the cartilaginous matrix. Addition of methylene blue to the growth medium lowered nitric oxide accumulation and prevented matrix degradation in the cultured cartilage discs. The cartilage matrix preservation effect was mediated through downregulation of all three isoforms of NOS, i.e., the neuronal NOS, endothelial NOS and inducible NOS and upregulation of TGF beta receptor in the chondrocytes. Our findings indicate that inhibition of NOS activity preserves cartilage matrix in vitro.

The epiphyseal organ contains two kinds of cartilage, articular and growth plate. Both enlarge during the growth phase of life. However, mitosis is not apparent in these tissues. In the current study, a search to trace the reservoirs of stem cells needed for the growth of these cartilages is done. A disorder in which the stem cells responsible for bone growth are mutated is achondroplasia; the mutation resides in the fibroblast growth factor receptor-3. Epiphyses stained with antifibroblast growth factor 3 antibodies reveal clusters of positively stained cells residing in the perichondrial mesenchyme, known as the ring of La Croix. Removal of the ring of La Croix causes a drastic growth arrest in the limbs of rat neonates. Cell cultures derived of the ring of La Croix biopsy specimens show high rates of cell proliferation and cell migration in vitro, in contrast to articular or growth plate derived chondrocytes. These cells stain intensely by antifibroblast growth factor receptor-3 antibodies and antiproliferative cells nuclear antigen, in contrast with articular and epiphyseal chondrocytes. Transfection of cells from the ring La Croix by an adenovirus vector containing the gene encoding for Escherichia coli beta-galactosidase (lacZ), allows tracing of these cells in tissues. Local injections were performed either to the ring of La Croix or to the joint cavity in a guinea pig model. A characteristic distribution was seen after injection. The transfected cells migrated to areas of bone and cartilage formation in the subchondral bone plate and on either side of the growth plate. This labeling and distribution is maintained for as many as 3 months after injection. The cells from the ring of La Croix appear to be responsible for bone growth. Furthermore, perichondrial cells and other precartilaginous cells expressing fibroblast growth factor-3 have been shown to be good cells for implantation to correct defects of articular cartilage.

Cartilage is exposed to mechanical loads, generating at the level of single chondrocytes a hyperosmotic stimulus (HOS). The direct effect of HOS on second messenger pathways in avian chondrocytes was evaluated by fluorimetric and image analysis techniques. HOS caused an immediate intracellular acidification of 0.07 +/- 0.02 pH units (n = 7), followed by an initial pH recovery rate of 0.033 +/- 0.04 pH units/min towards the pre-stimulus baseline values. Concomitantly, the intracellular calcium ([Ca2+]i) responded with a transient rise from baseline value of 84.7 +/- 7.4 nM to peak level of 403.1 +/- 51.0 nM (n = 16, p < 0.001). The calcium response was abolished by two calmodulin inhibitors chlorpromazine and W-7. Since these inhibitors are known to be specific ligands of a S-100 protein, its intracellular staining was determined following HOS. The amount of immunodetectable S-100 protein was significantly increased following exposure to HOS (p < 0.05), and did not require an increase of [Ca2+]i. It appears that compression of cartilage is transduced into HOS of chondrocytes, and further elicits its effects through transient intracellular elevation of protons and calcium ions accompanied by increased staining of S-100 protein.

The feasibility of using cartilaginous implants containing bone marrow derived chondrocytes in biological resurfacing procedures for correcting defects in articular cartilage was examined in goats. The experimental protocol included bone marrow aspiration, mesenchymal cell culturing, cell proliferation, favorable conditions inducing chondrogenic differentiation, and implantation of autogeneic and allogeneic cells. Autogeneic implant transplantations proved to be the best source for regeneration and repair of defective articular surfaces with use of densitometric computed image analysis of histochemical and immunohistochemical parameters on tissue sections. Allogeneic chondrocyte enriched cultures derived from bone marrow evoke a typical immune response in the host, expressed by the formation of fibrosis and progressive joint arthrosis. In the current study, a biological resurfacing procedure is described in detail for large mammals of similar weight and size as humans. Autogeneic mesenchymal cells derived from a bone marrow aspiration are the best cell source and when embedded in hyaluronic acid based adhesive glue make an excellent cartilaginous implant. The reparative regenerated cartilaginous tissue outcome within the defects appear different than neighboring normal articular cartilage shortly after surgery. Whether in the long term the cartilaginous remodeling process will shape the cartilage such that it more closely resembles the original articular cartilage is not known.

Background. Maffucci's syndrome (MS) is distinguished by the enigmatic association of benign cartilaginous bone tumors and soft tissue hemangiomas. Methods. This study was conducted to define the distribution of nerves and neuropeptides around these tumors. Results were measured by quantitative image analysis of immunohistochemical staining. Four types of tissues were compared: connective tissues around normal muscles, solitary hemangiomas, MS hemangiomas, and MS enchondromas (the last two from a single patient). Results. The number of nerves was found to be quadrupled in both types of hemangiomas as compared to normal connective tissue. A unique feature of MS tissues is the presence of an increased number of nerve fibers not only in the lesions but also in histologically normal margins of resection surrounding the lesions. Furthermore, hemangiomas of both types were found to contain a significantly higher number of calcitonin gene-related peptide-, substance P-, and methionine enkephalin-positive fibers than did normal muscle or its related fibroconnective tissue. These neuropeptides are mitogens, and their presence stimulates the growth of the abnormal blood vessels. Enchondroma fragments from an MS patient contained numerous methionine enkephalin-positive nerves. This neuropeptide is known to act as a growth factor in cartilage proliferation. Conclusions. A neural abnormality of the neuropeptidergic nervous system seems to relate to the abnormal tumors seen in MS.

The familial disease of hereditary multiple exostoses is characterized by abnormal skeletal deformities requiring extensive surgical procedures. In hereditary multiple exostoses patients there is a shortage in the pericellular glycosaminoglycan (GAG) of heparan sulfate (HS), related to defective activity of HS glycosyltransferases, mainly in the pericellular regions of chondrocytes. This study searched for a novel approach employing xylosides with different aglycone groups priming a variety of GAG chains, in attempting to alter the GAG compositional profile. Cell cultures of patients with osteochondroma responded to p-nitrophenyl β-D-xyloside by a significant increase in total GAG synthesis, expressed mainly in the extracellular domains, limited to chondroitin sulfate). The different β-D-xylosides, in addition to increasing the synthesis of extracellular GAGs, led to a significant depletion of the intracellular GAG domains. In mouse chondrocyte cultures, β-D-xylosides with different aglycones created a unique distribution of the GAG pools. Of special interest was the finding that the naphthalene methanol β-D-xyloside showed the highest absolute levels of HS-GAGs in both extracellular and intra-pericellular moieties compared with other β-D-xylosides and with controls without xyloside. In summary, β-D-xylosides can be utilized in chondrocyte cultures to modify the distribution of GAGs between the extracellular and intracellular compartments. In addition, xylosides may alter the profile of specific GAG chains in each moiety.

Among the objectives in writing the current chapter were the curiosity and the interest in allocating the sites and routes of migration of the reservoirs of the mesenchymal precartilaginous stem cells of the developing limbs in health and in disease. We chose to emphasize the events believed to initiate in these regions of stem cells, which may lead to growth retardation disorders. Thus, this narrow niche touches an enlarged scope of developmental biology angles and fields. The enclosed coverage sheds light on part of the musculoskeletal system, skeletogenesis, organogenesis of mobile structures and organs, the limbs, joints and digits (arthrology). It appears that the key role of the cartilage-bone regions is their responsibility to replenish the physis with committed chondrocytes, during the developmental, maturation and puberty periods. We shall start by outlining the framework of normal limb formation, the modalities, signals and the agents participating in this biological creation and regulation, illustrating potential sites that might deviate from normal development during the growth periods.

Stem cell development and fate decisions are dictated by the microenvironment in which the stem cell is embedded. Among the advanced goals of tissue engineering is the creation of a microenvironment that will support the maintenance and differentiation of the stem cell--based on embryonic and adult stem cells as potent, cellular sources--for a variety of clinical applications. This review discusses some of the approaches used to create regulatory and instructive microenvironments for the directed differentiation of mesenchymal stem cells (MSCs) using three-dimensional crystalline calcium carbonate biomaterials of marine origin combined with a hydrated gel based on hyaluronan.

1. Mechanical loading of cartilaginous tissue generates an increase in the concentration of cations in the extracellular matrix. This includes a decrease of the extracellular pH (pHo), which is known to affect the intracellular pH (pHi), thereby modifying the intracellular metabolism. Thus, the regulation of pHi is essential for the physiological function of cartilage. The fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF AM) was employed in order to assess the mechanisms responsible for control of the pHi in an embryonic avian chondrocyte cell suspension. 2. Steady-state pHi in the absence of physiological HCO3- was 7.15 +/- 0.01 pH units as compared to a pHi of 6.94 +/- 0.02 pH units in its presence (P < 0.01). The intrinsic buffering power of chondrocytes (beta i) was 38.9 mM/pH unit and the total buffering capacity (beta T) was 65.8 mM/pH unit. 3. Cells maintained in a Hepes-buffered solution were exposed to an intracellular acid load by the NH4+ prepulse technique (20 mM NH4Cl). The initial rate of pHi recovery was 0.106 pH units/min (n = 18). Amiloride (0.33 mM), an inhibitor of the Na(+)-H+ exchanger, or replacement of external sodium [Na+]o with choline induced a 60% inhibition of the recovery rate, indicating a predominant involvement of this antiporter in the response to intracellular acidification. 4. H(+)-ATPase inhibitors (oligomycin 20 micrograms/ml; N,N;-dicyclohexylcarbodiimide (DCC), 0.5 mM; N-ethylmaleimide (NEM), 0.25 mM) and iodomycin (2 mM), a metabolic cell suppressor, reduced acid extrusion by 25% as measured by the NH4Cl prepulse in Hepes-bathed cells. 5. Chondrocytes transferred from a Hepes-buffered solution to a 5% CO2-25 mM HCO3- medium (HCO3- solution) underwent a pHi decrease of approximately 0.20 pH units, followed by a regulatory alkalinizing response of 0.118 pH units/min. The Na(+)-H+ exchanger was responsible for only 15% of this alkalinization (amiloride, 0.33 mM), in contrast to its primary role in HCO(3-)-free solution. 6. The activity of a Na(+)-dependent Cl(-)-HCO3- exchanger in physiological HCO3- solution was estimated by addition of the inhibitors 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS; 0.5 mM) or diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; 100 microM) and by the suspensions of chondrocytes in a Na(+)-free solution. Acidification performed under these conditions resulted in a 45% inhibition of the recovery rate as compared to control rates.(ABSTRACT TRUNCATED AT 400 WORDS)

INTRODUCTION: Many disorders in clinical orthopedics present tissue degeneration and lack of repair due to lack of a scaffold., Chitosan is a sugar polymer that is produced from chitin., The current abstract describes our experience with chitosan as a tissue regeneration supporting scaffold. MATERIALS AND METHODS:, Several models were evaluated including the rat acute rotator cuff model, the lapine Masuda's disc degeneration model and, the medial menisectomy rat model., RESULTS: The use of chitosan enhances tendon repair in the rat rotator cuff model and prevents muscle atrophy., The use of chitosan gel appears to prevent disc degeneration in the masuda model., In the osteoarthritis model the use of chitosan leads to better cartilage preservation and less osteophyte formation., Animal pain is reduced as measured using an in capacitance meter. DISCUSSION:, The use of chitosan gel allows repair of several difficult animal models simulating common orthopedic maladies.

We read the paper by Trebicz-Geffen published in the April issue of the International Journal of Experimental Pathology with interest (Trebicz-Geffen et al. 2008). In their study, the authors reported molecular analysis of the multiple osteochondroma (MO)-associated EXT1 gene [MO (Bovee & Hogendoorn 2002) was previously known as hereditary multiple exostoses (HME) (Solomon 1964)], and documented their findings with mRNA and protein measurements, and with DNA sequence analysis. As these results are difficult to reconcile with aspects of current understanding of EXT1 function, and with previous studies on EXT1 and EXT2 expression in hereditary MO and solitary osteochondromas (SO), we would like to comment on their results in perspective. Their observation that EXT1 mRNA and protein levels are elevated in patients with MO who carry an EXT1 mutation was surprising. EXT1 has been shown to act as a tumour suppressor gene (Bovee et al. 1999; Bernard et al. 2001). In a subset (but not all) of MO patients, loss of the remaining wild type allele can be shown in the osteochondromas (Bovee et al. 1999). As 70% of the mutations found in EXT1 are non-sense frame shift or splice site mutations, leading to a premature stopcodon (Wuyts & Van Hul 2000), one would expect down-regulation at both mRNA and protein levels in MO. We reported that EXT1 and/or EXT2 mRNA levels are down-regulated in MO as compared with normal human growth plate (Hameetman et al. 2007a), and decreased levels of EXT1 and/or EXT2 protein in the cartilage cap cells of MOs have been reported previously (Bernard et al. 2001). In addition, their observation that normal EXT1 levels are present in SOs is surprising. Somatic mutations of EXT1 and EXT2 are very rare or absent in these tumours (Hecht et al. 1997, Hecht et al. 2002; Bernard et al. 2001). Recently, however, we have shown that homozygous deletion of EXT1 occurs in the cartilaginous cap of SOs (Hameetman et al. 2007b), suggesting that (similar to MOs) EXT1 is inactivated in SOs. These findings suggest that EXT1 mRNA and protein levels are likely to be down-regulated in both conditions, and this has been confirmed at the mRNA level (Hameetman et al. 2007a). The authors do not discuss these discrepancies. They propose that up-regulation of EXT1 is a compensation mechanism for the defective EXT1. This is difficult to reconcile with the alternative, which is that inactivation of EXT1 is causative for osteochondromas. An explanation for these conflicting data may lie in the different tissues that were used in the two studies. Our study used fresh frozen material from the cartilage cap, with confirmed cartilaginous morphology, whereas their study used cultured cartilage cap cells, without clarifying which markers were used to confirm this. It is therefore impossible to exclude that the cultured cells had a fibroblast-like phenotype without evidence about morphology and biochemistry. Furthermore, as control material, we used normal age-matched growth plate, and they used normal cartilage from patients undergoing total joint replacement. EXT1 mRNA expression is high in both human (Hameetman et al. 2007a) and mouse (Stickens et al. 2000) growth plate. Endogenous EXT1 mRNA levels have not been determined previously in human articular cartilage, and have not been documented in elderly people with severe osteoarthritis, which is the most likely source of material for their study. However, high EXT expression would not be expected, because proliferating and prehypertrophic chondrocytes (i.e. the cells that express EXT in the growth plate) are infrequent in articular cartilage. Moreover, postpuberty, one would expect low or absence of expression of EXT in human articular cartilage, as the downstream signalling targets of EXT, such as Wnt, transforming growth factor, and bone morphogenetic protein, are also altered to favour chondrocyte catabolic activity (Hopwood et al. 2007). The immunohistochemical staining of EXT1 in articular cartilage is difficult to evaluate but does not suggest high expression in figures 5 and 6 of the paper. Comparison to low EXT expression in articular cartilage will mean that there appears to be an increase in osteochondromas, whereas comparison with high endogenous EXT1 expression in growth plate cartilage will suggest down regulation. As osteochondromas are presumed to arise from growth plate cartilage (Hameetman et al. 2007b; Clement et al. 2008), we believe that this is the more relevant control. Another explanation, which could account for the difference in findings, may lie in the approach used for the Western blot analysis. Pre-immune sera showed different patterns in normal, SO and hereditary MO samples. The use of a standard more stably expressed internal control, such as alpha tubulin, could overcome this problem and might lead to different conclusions. Thus we suggest that the findings are difficult to interpret based upon current understanding on the function of EXT1, and on comparative expression (Bernard et al. 2001; Hameetman et al. 2007a), and that there are technical issues, including choice of positive controls, which might account for the observations reported.

Cell- and matrix-related parameters, which characterize the aging and differentiation process of cartilage in vivo, were measured in cultured chick epiphyseal chondrocytes during maintenance in a suspension culture for 34 days. A gradual decrease in the rates of proliferation and an increase in the size of the cells were observed. Ultrastructural examination revealed increased vacuolization and appearance of glycogen-storing pools. The rate of proteoglycan synthesis gradually increased. Age-related changes in the composition of the proteoglycan consisted of an increase in the ratio of keratan sulfate/chondroitin sulfate. The results indicate that the process of aging in culture resembles maturation and differentiation of cartilage tissue in vivo. The levels of cytosolic free calcium ions ([Ca2+]i) were measured in fura-2-loaded cells during the course of aging in culture. A gradual decrease in [Ca2+]i was observed. In 5-day cultures, a value of 184 nM [Ca2+]i was measured; this value decreased to 61 nM in 34-day cultures. On the basis of the present data and the previous results, which showed that cartilage-derived growth factors caused a decrease in [Ca2+]i, concomitantly with enhancing differentiation, whereas factors which elevated [Ca2+]i, caused an increase in proliferation and a decrease in proteoglycan synthesis, we suggest a model for control of chondrocyte differentiation and aging. The model suggests that the rate of differentiation may be paced by changes in steady-state levels of [Ca2+]i.

The potential of fresh whole chick epiphyses of embryonic origin to serve as implant material for cartilage defects of aged chicken was tested.Fresh epiphyses of 11-day-old embryos were collected from 24 animals and transplanted into defects created in the weight-bearing areas of tibiotarsal joint cartilage of 2-year-old chicks. Upon sacrifice, samples were examined macroscopically and microsections were prepared for histology.Macroscopically, control defects remained empty at all the time intervals. Defects of the experimental group were, on the other hand, filled with cartilaginous tissue as early as 2 weeks posttransplantation, although individual epiphyses could still be noted in the implant tissue. At 4 weeks and later, defects were filled with cartilaginous material indistinguishable from hyaline cartilage. Histologically, all grafts remained within the defect's pits, showing mitotic and metabolic activity typical to proliferating hyaline cartilage. The engrafted epiphyses showed a partial incorporation and integration with the surrounding host tissues already at 2 weeks. At 4 weeks and later, the integration was complete.It is concluded that a chick embryonic epiphyseal cartilage is suitable as a graft source for articular cartilage transplantation. The embryonic epiphyses provide immediate inherent stability to the graft and supply a good mix of mesenchymal progenitor cells responsible for the high rate of cell proliferation and adhesion to the differentiated committed chondrocytes of the host that create the typical favorable chondrogenic milieu. Based on the present findings, it is postulated that human embryonic epiphyses may, in the future, represent an alternative source to the commonly used techniques of hyaline cartilage repair.