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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.

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.

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.

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.

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.

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.

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.

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.

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.

Ample experimental examples have been accumulated during the last 3 decades indicating the ability of exogenous sources of cultured cells to serve as implants accelerating cartilage regeneration in defects of articular surfaces. In some cases, the repair tissues form complete spatial reconstruction of the defect. In other cases, either the spatial reconstruction is incomplete or the quality of the reparative tissue is inadequate. A delayed pace of endochondral ossification in the deep zones of the subchondral region of the defects, or ossification above the tide mark, within the superficial cartilaginous articular regions have been noted. Therefore, even in this promising approach of biological resurfacing procedure results are not certain, and further investigative research efforts are required. In the current study, a comparison of implantations of various cultured cells of four different sources were tested in an avian system. The reparative tissue outcomes are divided into three grades: full regeneration success, partial success, and failure of regeneration according to qualitative histological parameters and quantitative observation of the gross specimen. Defects that failed to regenerate a completely filled lesion were found to contain cells carrying the preskeletal–precartilaginous characteristic marker of FGFR3. The findings based on the above parameters suggest that autogeneic, chondrocytic-enriched bone marrow derived mesenchymal cells are superior to other cell sources for articular cartilage regeneration. Grafting of defects with these cells results in a 100% success rate. Allogeneic limb bud-derived mesenchymal cells and allogeneic embryonal chondrocytes have both reached a success of 75% of completely filled defects. Allogeneic chondrocytic-enriched bone marrow-derived mesenchymal cells yielded a 31% success rate. Untreated defects completely failed to heal. In successfully healed defects no cells of the reparative tissue carry the FGFR3 marker 3 months postimplantation. In partially healed defects, FGFR3 positive staining is present in fibrous cells at the invaginated surface. These latest findings may suggest some kind of proliferation failure in such cases.

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.

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)

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.

Background and Objectives Temperature-controlled tissue laser soldering is an innovative sutureless technique awaiting only solid experimental data to become the gold-standard surgical procedure for incision closure. The goals of the current study were: (1) to define the optimal laser soldering conditions, (2) to explore the immediate skin reparative healing events after sealing the wound, and (3) to determine the long-term trajectory of skin wound healing. Study Design/Materials and Methods Skin incisions were generated over rabbit dorsa and were closed using different wound-closure interventions, in three groups: (a) closure, using a temperature-controlled infrared fiberoptic CO2 laser system, employing 47% bovine serum albumin as a solder; (b) wound closure by cyanoacrylate glues; and (c) wound closure by sutures. The reparative outcomes were evaluated macroscopically and microscopically, employing semi-quantitative grading indices. Results Laser soldering of incisions at T = 65°C emerged as the optimal method achieving immediate wound sealing. This in turn induced accelerated reparative events characterized by a reduced inflammatory reaction, followed by minimal scarring and leading to a fine quality healing. Conclusions Temperature-controlled laser soldering offers an accelerated wound reparative process with numerous advantages over the conventional methods. Further investigations may reveal additional benefits in the spectrum of advantages that this innovative surgical technology has to offer. This can introduce new scientific insight that will pave the way for clinical use. Lasers Surg. Med. 35:1–11, 2004. © 2004 Wiley-Liss, Inc.

Combined modality treatment of musculoskeletal tumors led to improved patient survival. As survival improves, more consideration is given to the functional outcome of treatment, and interest is focused on the development of less mutilating and extensive surgery. One modality that can reduce patient disability significantly is cryosurgery, as it allows minimally invasive surgery based on marginal resection and tumor interface sterilization instead of wide resection of certain neoplasms. Classical cryosurgery as developed by Marcove involves pouring of liquid nitrogen into the tumor bed. This approach revolutionized the treatment of some tumors such as giant cell tumor of bone, allowing intra-lesional resection to substitute the wide-resection method used up to that time. However, complications of this method of treatment are common, including nitrogen emboli, fractures of the bone due to extensive necrosis and damage to neurovascular elements. A recent development in the field of cryosurgery has been the argon-based system allowing controlled formation of an ice-ball surrounding a metallic probe. The system is computer controlled and allows precise evaluation of the tumor bed interface as well as surrounding structures that need to be protected. Prior to application of this method in humans it is important to ensure that interface sterilization is indeed achieved using cryosurgery. To evaluate this question, a Swarm rat chondrosarcoma was used. Cell viability was assessed following ice-ball formation. Histological evaluation indicated that cell death occurs up to 5 millimeters from the ice-ball if temperatures of −40° Celsius at the metallic probe are achieved. A further evaluation was performed on samples obtained from patients during surgery. A minimum of two freezing cycles was shown to be necessary to achieve tissue viability similar to that of boiled tissue. Twenty-seven patients were operated to date using an argon-based cryosurgery system. The patients included 7 cases of grade I chondrosarcoma, 5 cases of giant cell tumor of bone, 14 cases of a metastatic lytic bone lesion and a single case of osseous-fibrous dysplasia. None of the patients suffered nerve injury during the operation. After a minimal follow-up period of 2 years only two of the surviving patients had a recurrence (a giant cell tumor of the proximal fibula, and the patient with the osseous-fibrous dysplasia whose tumor recurred as a frankly malignant adamantimoma). There were no pathological fractures. This method appears practical and allows close monitoring of the surrounding tissue to reduce the chances of recurrence.

This study was designed to assess a new composite implant to induce regeneration of injured spinal cord in paraplegic rats following complete cord transection. Neuronal xenogeneic cells from biopsies of adult nasal olfactory mucosa (NOM) of human origin, or spinal cords of human embryos, were cultured in two consecutive stages: stationary cultures in a viscous semi-solid gel (NVR-N-Gel) and in suspension on positively charged microcarriers (MCs). A tissue-engineered tubular scaffold, containing bundles of parallel nanofibers, was developed. Both the tube and the nanofibers were made of a biodegradable dextran sulphate–gelatin co-precipitate. The suturable scaffold anchored the implant at the site of injury and provided guidance for the regenerating axons. Implants of adult human NOM cells were implanted into eight rats, from which a 4 mm segment of the spinal cord had been completely removed. Another four rats whose spinal cords had also been transected were implanted with a composite implant of cultured human embryonic spinal cord cells. Eight other cord-transected rats served as a control group. Physiological and behavioral analysis, performed 3 months after implantation, revealed partial recovery of function in one or two limbs in three out of eight animals of the NOM implanted group and in all the four rats that were implanted with cultured human embryonic spinal cord cells. Animals of the control group remained completely paralyzed and did not show transmission of stimuli to the brain. The utilization of an innovative composite implant to bridge a gap resulting from the transection and removal of a 4 mm spinal cord segment shows promise, suggesting the feasibility of this approach for partial reconstruction of spinal cord lesions. Such an implant may serve as a vital bridging station in acute and chronic cases of paraplegia.

This study compared the articular cartilage repair potential of cultured chondrocytes transplantation with bone-cartilage paste-graft in the resurfacing of full-thickness defects without breaching of the subchondral bone plate in rabbit knees. A 5 x 5-mm articular cartilage defect was created in the patellar groove of the femur. Three months following creation, the defect was filled with cultured autologous chondrocytes (group 1) or bone-cartilage paste (group 2). A control group of untreated defects was followed for 1 year. The reparative tissue was analyzed macroscopically, histologically, and by immunohistochemistry 3-12 months post-transplantation. The surfaces of the reparative tissue in group 1 were smooth, and the defects were filled with reparative tissue that resembled hyaline cartilage. The composition of the repair tissue more closely resembled cartilage, as demonstrated by cartilage-specific stains. In contrast, the reparative tissue in group 2 was fibrous and exhibited markers of mesenchymal stem cells and bone formation. Transplantation of cultured chondrocytes into a full-thickness defect in the rabbit generates a biologic substitute tissue that resembles native articular cartilage with living cells capable of synthesizing the surrounding cartilage matrix. In contrast, analysis of the healing response to the paste-graft technique failed to show cartilage-like characteristics. This information may be clinically applicable to direct the use of these treatments in chondral injuries.

This pilot study examined the effects of composite implants of cultured embryonal nerve cells and laser irradiation on the regeneration and repair of the completely transected spinal cord. Embryonal spinal cord nerve cells dissociated from rat fetuses and cultured on biodegradable microcarriers and embedded in hyaluronic acid were implanted in the completely transected spinal cords of 24 adult rats. For 14 consecutive post-operative days, 15 rats underwent low power laser irradiation (780 nm, 250 mW), 30 min daily. Eleven of the 15 (73%) showed different degrees of active leg movements and gait performance, compared to 4 (44%) of the 9 rats with implantation alone. In a controlgroup of seven rats with spinal cord transection and no transplantation or laser, six (86%) remained completely paralyzed. Three months after transection, implantation and laser irradiation, SSEPs were elicited in 69% of rats (p = 0.0237) compared to 37.5% in the nonirradiated group. The control group had no SSEPs response. Intensive axonal sprouting occurred in the group with implantation and laser. In the control group, the transected area contained proliferating fibroblasts and blood capillaries only. This suggests: 1. These in vitro composite implants are a regenerative and reparative source for reconstructing the transected spinal cord. 2. Post-operative low power laser irradiation enhances axonal sprouting and spinal cord repair.

Osteogenic growth peptide, a histone H4-related, 14-amino-acid peptide, is an active mediator of local, as well as systemic, osteogenic activity in response to marrow ablation, trauma, and blood loss. In this study, the effect of exogenous osteogenic growth peptide on the healing of femoral fractures in rats was investigated. A fracture at the midshaft of the femur was created in 50 rats. Half of the rats were injected subcutaneously with 25 ng of osteogenic growth peptide per rat per day for the first 7 days after fracture. Radiographs were taken each week, and the diameter of the callus was measured. The femurs of four animals from each group were harvested 1, 2, 3, and 4 weeks after fracture. Two femurs from each group were sectioned for histologic examination, and two were sectioned for measurement of density and mineral content. Marrow was aspirated from the contralateral femurs to establish adhering cell cultures, which were examined for osteogenicity. At 2 weeks, a large increase in mitogenicity and osteogenicity was seen in the marrow-derived cultures from the rats treated with osteogenic growth peptide; this increase was sustained through 4 weeks. Extraction of RNA from the contralateral marrow (systemic expression) and callus (local expression) for amplification with reverse transcription-polymerase chain reaction revealed greater systemic expression of transforming growth factors beta1, beta2, and beta3, fibroblast growth factor-2, insulin-like growth factor-1, and aggrecan throughout the 4 weeks after fracture, whereas types IIA and IIB collagen, link protein, and fibroblast growth factor receptor-3 had a greater local expression. The specimens treated with osteogenic growth peptide had a stronger expression of transforming growth factor-beta1, both locally and systemically. The average diameter of the callus was greater for the treated rats at all time intervals, and peak diameters were 7.58 mm at 3 weeks for the treated rats and 6.64 mm at 2 weeks and 6.63 mm at 3 weeks for the controls. Histological study revealed an earlier organization and faster healing of the treated fractures, as evidenced by the larger, earlier appearance of cartilaginous soft callus and the more rapid organization of bridging trabecular bone. No statistical significance was obtained when these comparisons were made between the groups. These results suggest that osteogenic growth peptide can be used to promote earlier proliferation and differentiation of osteogenic cells in marrow and bone-repair callus, possibly through its effect on the transforming growth factor-beta family.

During the prenatal period, gonadal steriod environment induces dramatic sexually dimorphic changes in the nervous system. We have used in vitro methods to study the mechanism and timing of hormonal influences on neuronal sprouting and myelination during the prenatal period. Organotypic cultures of hypothalamus and lumbar spinal cord (SC) slices from rat fetuses were grown on plasma clot or in hyaluronic acid and exposed to estogen (17β estradiol) and testosterone (T) during cultivation. Both steriod hormones were active: 17β estradiol enhanced sprouting of hypothalamic neuronal fibers and increased the amount of synapses. In SC cultures T induced regeneration of thick nerve processes and an early onset of myelination, mainly of peripheral myelin.

1. The effects of hyperosmotic stress on cytosolic calcium concentration ([Ca2+]i) were studied by ratio image analysis in single cells of an osteoblast-like bone cell line (RCJ 1.20) loaded with fura-2 AM. 2. The ratio (340 nm/380 nm) of steady-state [Ca2+]i in resting osteoblasts kept in Hepes-buffered medium was 0.82 +/- 0.04. A hyperosmotic stimulus (200 mosmol l-1 sucrose) produced a [Ca2+]i transient with a peak ratio of 1.28 +/- 0.09, which decayed with an apparent half-life (t1/2) of 42.7 +/- 2.6 s. 3. The hyperosmotically induced [Ca2+]i transients were insensitive to verapamil, diltiazem or nifedipine, which excludes the involvement of dihydropyridine-sensitive Ca2+ channels in the process. Non-specific Ca2+ channel blockers (Mn2+, Ni2+, La3+ or Gd3+) partially abolished the hyperosmotically induced [Ca2+]i elevation, indicating the contribution of extracellular Ca2+ influx. 4. A hyperosmotic stimulus applied in Ca(2+)-free medium (0.5 mM EGTA) lowered the [Ca2+]i peak to a ratio of 0.96 +/- 0.08 (P < 0.001) compared with a Ca(2+)-containing medium. This suggests that the [Ca2+]i increase is due to extracellular influx, as well as release from an intracellular Ca2+ pool. 5. Application of thapsigargin (0.5 microM), a specific inhibitor of endoplasmic reticulum Ca(2+)-ATPase, in Ca(2+)-free medium caused transient [Ca2+]i elevation to peak ratios of 1.33 +/- 0.09, and completely abolished the [Ca2+]i response to a hyperosmotic stimulus. This implies the existence of a thapsigargin-sensitive intracellular pool of Ca2+ that is mobilized by hyperosmotic stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple, linear polysaccharide with unique molecular functions, hyaluronan is a glycosaminoglycan whose biomechanical and hydrodynamic properties have been thoroughly characterized. However, the exact role the molecular mechanisms and signaling pathways of hyaluronan play in the regulation of stem cell fate, such as self-renewal and differentiation, remains to be determined. The abundance of hyaluronan in embryonic tissues indicates that it is highly important in developmental processes. Recent studies have focused on understanding the mechanisms of hydrated hyaluronan action and its interaction with neighboring substances. This review is an attempt to elucidate the complex role of hyaluronan signaling in the initialization and regulation of developmental processes, particularly in events dictating the fates of mesenchymal stem cells during the organogenetic phases of chondrogenesis and osteogenesis.

Cartilaginous fetal bones from rat preserved by deep freezing procedures were compared to comparable fresh bones with regard to the following parameters: chemical composition, water and uronic acid contents; cell viability measured by the rate of proteoglycan synthesis; mineralization-ossification status by calcium binding; matrix integrity by the release of uronic acid containing substances; and biological activity as transplants inducing the formation of bone. The transplanted material was chemically analyzed and checked for its rate of proteoglycan synthesis. The quality of the formed bone was similar whether isogeneic or allogeneic, fresh or cryopreserved bone was employed as transplant material. Evidently those various fetal bones may be of clinical value whenever the need for replacement of massive bone loss arises. Although the viability and the cartilaginous nature of the graft are critical, the isogeneity and freshness are of a quantitative advantage only. These biochemical observations were confirmed by roentgenological and histological evaluations of the grafts. An optimal cryopreserving procedure and tests for examining bone candidates for successful grafting are described.

The activity of UDP-xylose:core protein xylosyltransferase (EC 2.4.2.26) in costal cartilage of young rats (3 months) and old rats (36 months) was measured. The enzyme activity in cartilage of young rats (mean +/- S.D.) is 3370 +/- 1440 Bq h-1 mg-1 DNA, which is about three times higher than that determined in cartilage of old rats (1090 +/- 520 Bq h-1 mg-1 protein). The amount of galactosamine-containing proteoglycosaminoglycans that are extractable with 4 M guanidinium chloride from cartilage is significantly higher in young rats (29.1 +/- 4.8 nmol GalN per mg cartilage wet weight) than in old animals (5.8 +/- 3.0 nmol GalN per mg cartilage wet weight). Thus, if xylosyltransferase activity is referred to the amount of galactosamine-containing proteoglycans in cartilage, nearly identical values are obtained (young rats, 80 +/- 30 Bq h-1 mumol-1 GalN; old rats, 85 +/- 35 Bq h-1 mumol-1 GalN). The results support the assumption that the synthesis of proteochondroitin sulfate is diminished in costal cartilage of old rats by a mechanism involving a reduced activity of xylosyltransferase.

Embryonic chick pelvic cartilages were incubated in the presence of insulin like growth factor (IGF) (1–100 μU/ml), as well as normal human serum (5%), with radiolabelled precursors of proteoglycan (PG) synthesis: L-[3-3H]serine, D-[6-3H]glucosamine and [35S]Na2SO4. IGF alone (1–15 μU/ml), stimulated in a dose-dependent manner D-[6-3H]glucosamine incorporation into tissue-bound and soluble isolated glycosaminoglycan (GAG) chains. L-[3-3H]serine incorporation into PG molecules was not stimulated by IGF (1–100 μU/ml), despite the increase in the uptake of this precursor into intact cartilage. [35S]Na2SO4 incorporation was unaffected by IGF. Serum promoted the uptake of all three precursors into tissue-bound glycosaminoglycans. It was postulated that IGF could stimulate proteoglycan synthesis not only by elongating existing chondroitin sulphate chains but also by increased synthesis of other sugar chains e.g. keratan sulphate and oligosaccharides.

The effects of normal human serum, insulin-like growth factor and β- d -xyloside on the synthesis of proteoglycan, as well as their differential effect on the synthesis of chondroitin sulfate and keratan sulfate side-chains, were studied in chick embryonal cartilage. The glycosaminoglycans found in the incubation medium were mainly intact carbohydrate moieties of partially degraded proteoglycan molecules, whereas the tissue-bound glycosaminoglycans were of intact proteoglycan molecules. In incubations with normal human serum, the synthesis of the chondroitin sulfate side-chains of the tissue-bound glycosaminoglycans was preferentially stimulated, while the percentage of medium glycosaminoglycan (out of the total glycosaminoglycan in tissue and medium) was reduced, compared to control incubations. In incubations with insulin-like growth factor, the synthesis of the keratan sulfate side-chains of the tissue-bound glycosaminoglycan was preferentially stimulated, whereas the percentage of the medium glycosaminoglycan resembled that of control incubations. In incubations with xyloside, a marked reduction of tissue-bound glycosaminoglycan was noticed, mainly of chondroitin sulfate chains, and only a slight decrease in keratan sulfate chains. Human serum of various age groups stimulated proteoglycan synthesis in embryonal chick cartilage to almost the same extent. However, sera from babies and adults were found to stimulate chondroitin sulfate chains preferentially, whereas serum of aged subjects preferentially enhanced the synthesis of keratan sulfate chains. These findings suggest that the synthesis and/or degradation of the various types of glycosaminoglycan chains (chondroitin sulfate and keratan sulfate) of cartilage proteoglycan can be regulated differentially by serum growth factors. Secondly, the growth hormone-mediated serum factor (insulin-like growth factor) seems to play a role in molecular aging of proteoglycans.

Only a very small portion (4-7%) of the hyaluronidase of rat sperm obtained from the caput or cauda epididymis was related during incubation in capacitation medium for up to 24 h at 37 degree C. A portion of the cell-associated hyaluronidase was accessible to external substrates, allowing sperm suspensions to lyse glycosaminoglycans of cumulus clots rapidly, even without capacitation. This process appears to represent the employment of an enzyme bound to the sperm cell, analogous to the use of solid-phase enzymes in current enzyme technology.

The pleiotypic effect of somatomedins on cells is well-known. This review deals with somatomedins (SMs) as regulators of proteoglycan (PG) synthesis. Once the mechanism of this role of SMs is understood, it can be used as a general macromolecular model for studying the effects of SMs on matrix components of connective tissues. The factors regulating PG production rate can be classified into two categories: 1. Intracellular factors which integrate the following: the genetic code, the synthetic enzymes and their organization, the availability of building block substances, various cell nutritional factors, and =-degrading enzymes. All these determine the basic rate of production. 2. Extracellular factors, including hormones secreted from glandular organs and tissuelocal agents with regulation ability. The hormonal factors may alter rates of PG production and generate differences in the product composition, reflecting changes in the individual’s developmental, physiological and/or pathological status. While hormones such as thyroxine which stimulates the rate and glucocorticosteroids which suppress the rate have been reported to regulate PG synthesis, the somatomedin (SM) family seems to play a key role in this regulation. The term somatomedin denotes the relation of a substance to somatotrophin (growth hormone (GH)), and its intermediary (mediator) role in transmitting the message of GH to the target or soma cells (e.g. fibroblasts, chondrocytes, osteocytes, etc.). At most, the SM family includes six members: SM-A, SM-B, SM-C, NSILA (non-suppressible insulinlike activity) I and 11, or in the new terminology IGF (insulin-like growth factor) I and 11, and MSA (multiplication stimulating activity). The SMs share certain common elements, and some of their characteristics are listed in Table I. In brief, the SMs are a group of low molecular weight polypeptides (ranging from 4000 to 8000 daltons) whose biological activity can be expressed as growth-promoting , insulin-like and mitogenic. The growth-promoting activity is assessed mainly by the effect of SM on the rate of matrix production: this is measured as the stimulation of sulfate incorporation into cartilage slices of various animals. The endocrine route of SMs begins with the hypothalamic secretions which cause the release of GH from the pituitary. This GH is believed to generate the synthesis of SMs, mainly in the liver and kidney, which then operate on their target cells via specific receptor sites on the cell membranes. PG synthesis (matrix production) has become over the years the indicator of the overall growth process of the bony framework and the level of SMs in blood has come to serve as the clinical anabolic index of serum content in growth factors.

Chondrocytes in suspension culture preserve their differentiated state, as expressed by a high rate of chondromucoprotein synthesis. Purified chondromuco-protein added to a suspension of cultured embryonic epiphyseal cartilage cells stimulates the rate of synthesis of chondromucoprotein at concentrations up to 2 mg/ml. The stimulation involved synthesis of the entire molecule since incorporation of labeled serine (into core protein), glucosamine, acetate, or sulfate was increased. Neither the amount of synthesis of total protein nor that of collagen was increased. No elevation of xylosyltransferase and N -acetylgalactosaminyltransferase activities was observed. The stimulation was produced by several other polyanionic substances. No evidence was found of the entry of chondromucoprotein into chondrocytes under the conditions of the experiments. Mild trypsinization lowered the response to added chondromucoprotein and decreased the endogenous rate of chondromucoprotein synthesis. Incubation of cells after trypsinization results in restoration of the rates of chondromucoprotein synthesis. The endogenous, as well as the stimulated, rate of chondromucoprotein synthesis varied with the density of chondrocytes in culture. It is suggested that the rate of chondromucoprotein synthesis may be partially controlled by cell surface receptors.

Embryonic chick chondrocytes growing in suspension culture excrete into the medium chondromucoprotein which appears to be identical to chondromucoprotein isolated from epiphyseal cartilage. Low oxygen tension favored maximum production of chondromucoprotein. The presence of ascorbic acid enhanced both chondromucoprotein and collagen synthesis. Glycosyltransferases involved in chondromucoprotein synthesis were detected in homogenates of such cultured chondrocytes.

Activation of the Na+/H+ exchanger following isosmotic and hyperosmotic stimuli was investigated in an osteoblast cell line (RCJ 1.20). The pH dependence of the transporter activity was studied under conditions of abolished proton gradient (pHi = pHo) across the membrane. The isotonic response is Na+o dependent, increases towards higher pH-values, displaying a sigmoidal dependence on pHi = o (Hill coefficient approximately 1.8) and is controlled by pHo. The greater than first order dependence on pH suggests that H+o inhibits the exchange beyond the rate expected from competition with the Na+o alone. This may be due to the existence of an external H+ regulatory site with a negative cooperative effect on the intra- or extracellular transport site. The hyperosmotic activation is Na+o independent, parallels the sigmoidal pH dependence of the isosmotic stimulus (Hill coefficient approximately 2.0) and is mediated through an increase of the Vmax without a change in the intracellular proton sensitivity.

Cultured epiphyseal-chondrocytes from embryonic chick may serve as a useful in vitro model to study aging processes in cartilage. The accelerated aging process in cultured chondrocytes is completed within a month and is manifested by typical changes in both cellular and extracellular compartments. Under common maintenance conditions, cells show a gradual loss of replicative capacity, increase in the rate of proteoglycan synthesis and age-dependent changes in the structure and composition of proteoglycan. An environmental factor — reduced oxygen tension — was found to slow down aging processes and preserve the young features of chondrocytes for a longer duration in culture. Cultures maintained under lower oxygen tension had higher proliferation rate, smaller cell size, lower rate of proteoglycan synthesis, and lower content of keratan sulfate side chains in the proteoglycan. In addition higher concentrations of free cytosolic calcium [Ca 2+ ] in as compared to control cultures, was found. It is suggested that the increased proliferation rate and the decrease in proteoglycan synthesis caused by low oxygen tension may be signalled by the higher [Ca 2+ ] in in these cells.

Chondrocytes derived from chick embryos can be successfully implanted in defects of adult chick articular cartilage surfaces. Such implants thrive in their implantation site and create a new articular surface. The chondrocytes mature and hypertrophy in the orthotopic site without invoking an immune response. Eventually a steady state is reached in which mature chondrocytes resurface the defect while in the deeper areas spongy bone replaces the hypertrophic chondrocytes. Time schedules of these repair events have been studied in hosts of different ages. We compared 4-month-old chicks with 3-year-old chickens. The embryonal chondrocytes implanted in the latter group underwent an accelerated aging process. The defects were completely filled-up after 1 month as compared with 2–3months in the younger age group. Endochondral ossification in the older group was evident as early as 2 months post implantation and was completed after 6 months. This contrasts with the situation in the younger group where the chondrocytes only began to hypertrophy after 6 months. At this stage endochondral ossification was hardly seen at all. A unique response to the cartilaginous implants is seen in the old group only in the vicinity of the reparative tissue, accumulation of hematopoietic centers. This study seem to indicate that the host's environment affects the “biological clock”, i.e. rate and degree of aging of the implanted cells, as well as their matrices.

The nature of the proteoglycan(s) (PG) found in the extracellular matrix (ECM) layer produced by cultured bovine corneal endothelial (BCE) cells is analyzed. The PG(s) account for approximately 5 to 6% of the dry weight of the ECM, regardless of the amount of extracellular soluble PG available in the medium. A 4 M guanidinium chloride (GuCl) extract of ECM was separated on a dissociative cesium chloride (CsCl) gradient (1.25 g/cm3 starting density). Results showed one main peak of PG substance(s) comprising 91% of the total labelled substance and uronic acid, banding at a specific buoyant density of 1.29 g/cm3. The molecular weight of this major PG(s) as estimated by gel filtration on Sepharose CL-4B ranged from 0.5 to 0.7 X 10(6). Further chemical analysis of the main PG(s) band revealed a protein moiety accounting for 45% of the weight and carbohydrates-glycosaminoglycans (GAG) accounting for the remaining 55%. Analysis of the GAG chains (over the entire gradient) showed a composition, based on the susceptibility of the PG substance(s) to degrading enzymes, of 50% heparan sulfate, 43.5% dermatan sulfate, and 6.5% chondroitin 4- and 6-sulfate chains. BCE cell cultures grown in the presence of beta-D-xyloside produced an ECM lacking more than 90% of the GAG content found in the control ECM. The medium-soluble GAG chains, produced in vast excess in cultures grown in the presence of beta-D-xyloside, are composed mainly of chondroitin 4- and 6-sulfates.

In a previous study we showed that allografts of BN fetal bone, unlike allografts of adult bone, are not rejected by allogeneic recipients of the Lewis strain in spite of the existence of major histocompatibility complex (MHC) incompatibility between donors and hosts. In the present study, we analyzed the relationships existing between the host and fetal tissue that determine graft survival. We found that (1) the fetal BN graft, unlike adult grafts, induces in Lewis recipients a vigorous humoral response consisting mainly in the production of IgG antibody that seems to be directed against antigens of Ia-like specificities. (2) The BN rats are genetically defective in their capacity to respond to determinants and thus are not capable of producing anti-Ia antibodies; in accordance, Lewis fetal bone grafts are rejected by the BN recipients. (3) Chondrocytes isolated from fetal mouse bones do express Ia antigenic determinants. We suggest that the survival of an allogeneic fetal graft in an immunologically intact recipient depends on an active and selective immune response directed against the Ia components associated with the MHC on the embryonic and fetal cells. On the basis of these notions, we propose that the capacity of Ia determinants expressed on cells of the embryo, to elicit anti-Ia and IgG alloantibodies in the pregnant mother, determines the capacity of the embryo to escape rejection by the histoincompatible mother.

Cartilage-derived growth factors, enhance proteglycan synthesis in cultured chick-embryo chondrocytes, and have almost no effect on cell proliferation. Addition of carilage derived growth factors to cartilage cells loaded with the fluorescent Ca 2+ indicator quin 2, caused a rapid, concentration dependent decrease in cytoplasmic free Ca 2+ . This decrease persisted also in Ca 2+ -free medium, indicating that it is not mediated by a decrease in the passive permeability of cell membrane to Ca 2+ . Addition of the Ca 2+ ionophore A23187, with or without cartilage derived factors, caused an increase in cytoplasmic free Ca 2+ together with inhibition of proteoglycan synthesis and enhanced cell proliferation. The results may indicate that whereas cell proliferation in chondrocytes is signaled by an increase in cytoplasmic Ca 2+ ([Ca 2+ ] in ), proteoglycan synthesis is signaled by a decrease in [Ca 2+ ] in . The data lead to suggesting a mechanism for antagonistic regulation of cell proliferation and the expression of the differentiated state.

Circulating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells (B16-BL6 melanoma; ESb T-lymphoma) attach, invade, and penetrate confluent vascular endothelial cell monlayer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the [35S]O4 = -labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The macrophages do not store the heparanase intracellularly but it is instead found pericellularly and requires a continuous cell-matrix contact at the optimal pH for maintaining cell growth. The degradation of [35S]O4 = -labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10 micrograms/ml), arteparon (10 micrograms/ml), and heparin at a concentration of 3 micrograms/ml. In contrast, other glycosaminoglycans such as hyaluronic acid, dermatan sulfate, and chondroitin sulfate as well as the specific inhibitor of exo-beta-glucuronidase D-saccharic acid 1,4-lactone failed to inhibit the degradation of sulfated proteoglycans in the subendothelial extracellular matrix. Degradation of this heparan sulfate proteoglycan is a two-step sequential process involving protease activity followed by heparanase activity. However, the following antiproteases--alpha 2-macroglobulin, antithrombin III, leupeptin, and phenylmethylsulfony fluoride (PMSF)--failed to inhibit this degradation process, and only alpha 1-antitrypsin inhibited the heparanase activity. B16-BL6 metastatic melanoma cell heparanase, which is also a cell-associated enzyme, was inhibited by heparin to the same extent as the macrophage heparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase, inhibited at concentrations of 1 and 3 micrograms/ml, respectively. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood-borne tumor metastasis.