Development of a tissue-engineered composite implant for treating traumatic paraplegia in rats.

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.

Mechanical resistance of biological repair cartilage: comparative in vivo tests of different surgical repair procedures.

The former common knowledge that cartilage lesions do not heal has been modified over the last few years due to new technologies. For repair of deeper circumscribed lesions osteochondral press-fit grafting and tissue engineering are used in clinical application. The histological data of the hyaline-like tissue obtained by engineering are just as satisfactory as the surviving grafted hyaline cartilage on top of osteochondral cylinders. But comparative studies are still lacking. To fill the gap and with a view to repairing larger osteoarthritic defects we have performed an in vivo study on 16 goats. Three months after the creation of a full thickness wide cartilage defect on the femoral condyle with harvesting of cartilage samples for tissue cultures we performed secondary cartilage repair procedures on the installed osteoarthritis areas: 1) grafting with autogenic osteochondral press-fit cylinders from the opposite knee, 2) autologous engineered chondrocyte grafting under periosteal flaps, 3) both in combination. The harvesting defects were either left as controls or filled with a hyaluronate fleece. After eight months the repaired areas and the harvesting defects were examined for cartilage stiffness as a novel comparative parameter. Compared to normal the cartilage on top of osteochondral grafts is considerably stiffer. Engineered cartilage is weaker than normal. Spontaneously ingrown fibrous cartilage is much weaker even with a carrier fleece. A combination of osteochondral press-fit grafts with engineered autologous cells restores biomechanical qualities to repaired larger degenerative cartilage defects.

Role of FGF9 and FGF receptor 3 in osteochondroma formation.

Osteochondromas are chondro-osseous protuberances that occur in metaphyses of long bones. The cartilaginous cap is assumed to be responsible for the growth of the lesions during childhood and adolescence, but mitotic figures are rarely seen in the cap. Therefore, another cell population, probably mesenchymal cells, is responsible for proliferation and growth. Residual mesenchymal cells capable of rapid proliferation are difficult to detect due to lack of specific histologic features. Two specific markers for mesenchymal cells, FGF receptor 3 (FGFR3) and collagen type IIa, have been described. Osteochondroma mesenchymal cells are found in the soft tissues overlying the cartilage cap. The surrounding areas of typical cartilage are negative for both mesenchymal cell associated antigens. The soft tissues overlying the cartilage do not have cartilaginous features. The undifferentiated cells overlying the exostosis yield in culture a rapidly proliferating homogenous population of fibroblast-like cells. Expression at the mRNA level of FGF9, FGFR3, and collagen type IIa is found in these cells, but not in skin fibroblasts from afflicted or healthy individuals. Exogenous administration of TGFbeta1 to cultures of hereditary multiple exostosis eliminates FGF9 expression. These results indicate fibrous regions contain the mesenchymal cells responsible for osteochondroma growth.

Characteristics of cartilage biopsies used for autologous chondrocytes transplantation.

Biopsies removed from 57 patients considered for cartilage transplantation were grown at CTI Ltd. (47 biopsies) and at Tel Aviv University (10 biopsies). Tissue processing took place in dedicated laboratories. Explant cultures allowed cell number expansion. Fifty-four out of 57 biopsies grew cells. Fanning out of the cells began after 5-15 days in culture. Two passages later, cell numbers in the 10(7) range were achieved. Cells from all cultures expressed mRNA of aggrecan and link protein but not of alkaline phosphatase. Histochemical stains such as alcian blue pH 1 were negative in sparse monolayer cultures, but positive in pellet cultures. Immunohistochemistry demonstrated expression of collagen type I in monolayer cultures, switching to collagen type II in micromass cultures. Fibroblast growth factor receptor 3, a recently described characteristic receptor of precartilaginous cells, was expressed in monolayers and disappeared in micromass cultures. In conclusion, explants of articular chondrocytes cultured in vitro consistently yield monolayer cultures. The cells appear to revert to dedifferentiated chondrocytes, expressing a mesenchymal stem cell protein profile. Simultaneously, these cells regained their capacity to proliferate. Cultures held as micromass allowed reexpression of the differentiated phenotype traits.

Two freezing cycles ensure interface sterilization by cryosurgery during bone tumor resection.

Cryosurgery utilizing an argon-based system allows bone-tumor interface sterilization, while avoiding the risks of conventional cryosurgery. This study was conducted in order to evaluate the number of freezing cycles required for interface sterilization in cases of aggressive human bone tumors. Sixteen tumors were included (six chondrosarcomas, eight metastatic carcinomas, and two giant cell tumors). All occurred within long bones. In all cases a standardized marginal resection was performed. Following thorough curettage, we sampled five different locations within the tumor interface by a cylindrical hollow trephine. The interface viability was assessed using the XTT method. Quantitative histological evaluation was based on the percentage of live cells divided by total lacunae number in five random medium-power fields. One freezing cycle (5 min, -40 degrees C) reduced tumor viability to approximately 5% of prefreezing. However, there were still live specimens. Two or three freezing cycles led to complete interface sterilization. The difference between a single freezing cycle and two freezing cycles was significant (ANOVA, F = 130, P < 0.01). The difference between two freezing cycles and three freezing cycles was not significant (ANOVA, F = 0.14, P < 0.6). The results of the XTT method for the assessment of interface viability correlated well with histological evaluation of the percentage of viable cells (r = 0.89), as well as with cell culture results of frozen vs. prefreezing tumor samples. In conclusion, two freezing cycles are adequate to achieve tumor-bone interface sterilization in aggressive human bone tumors.

Articular Cartilage Chondrocytes are more Advantageous for Generating Hyaline-like Cartilage than Mesenchymal Cells Isolated from Microfracture Repairs.

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.

Storing live embryonic and adult human cartilage grafts for transplantation using a joint simulating device.

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. Avoidance of freezing of cartilage grafts, might prevent the cartilage degeneration often observed in frozen osteochondral grafts.

Synovial chondromatosis: the possible role of FGF 9 and FGF receptor 3 in its pathology.

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.

[Autologous chondrocyte transplantation--from science fiction to routine clinical practice].

Adult articular cartilage lacks the capacity for self-repair. The limiting factor appears to be the inability of chondrocytes to proliferate while embedded in the extracellular matrix typical of hyaline cartilage. Cartilage defects larger than 1 cm2 change articular biomechanics and lead to eventual osteoarthritis and joint destruction. During the past decade, several competing techniques have evolved to stimulate articular cartilage repair. Small lesions can be successfully treated by either micro-fracture or osteochondral cylinder grafting. The latter technique allows immediate weight bearing but leads to damage of previously uninvolved areas of articular cartilage, which limits its application to lesions of less than 2 cm2. When the damaged area is more extensive, grafting of autologous chondrocytes should be considered. First a diagnostic arthroscopy is performed to assess the damaged area and a small cartilage biopsy is taken. 6 weeks later, arthrotomy and chondrocyte transplantation are performed. In the interval, the antologous chondrocytes have expanded by 2 to 3 orders of magnitude. Our experience to date includes 10 cases with follow-up of 6 months to 5 years. Preoperative complaints of crepitation and locking disappear. There is functional improvement and pain reduction of approximately 50%. This procedure, currently limited to patients under 55 years of age with limited damage to an articular surface, for the first time allows reconstruction of damaged articular areas without resorting to allografts.

Autologous chondrocyte transplantation for reconstruction of isolated joint defects: the Assaf Harofeh experience.

BACKGROUND: Articular cartilage is incapable of undergoing self-repair since chondrocytes lose their mitotic ability as early as the first year of life. Defects in articular cartilage, especially in weight-bearing joints, will predictably deteriorate toward osteoarthritis. No method has been found to prevent this deterioration. Drilling of the subchondral bone can lead to fibrocartilage formation and temporary repair that slowly degrades. Animal experiments indicate that introducing proliferating chondrocytes such as cultured articular chondrocytes can reliably reconstruct joint defects. OBJECTIVES: To describe our clinical experience in culturing and transplanting autologous chondrocytes. METHODS: Biopsies were obtained from 10 patients, aged 18-45, undergoing a routine arthroscopy in which a cartilage defect was identified with indications for cartilage transplantation. The biopsies were further processed to establish chondrocyte cultures. ACT was performed in 8 of the 10 patients because of persistent symptoms for at least 2 months post-arthroscopy. All patients (6 men and 2 women) had a grade IV cartilage defect in the medial or lateral femoral condyle, and three had a defect in the trochlear region as well. Biopsies were removed from the lateral rim of the superior aspect of the femur, and cells were cultured in a clean room. Following a 2 order of magnitude expansion, cells were implanted under a periosteal flap. RESULTS: The eight patients implanted with autologous cells were followed for 6 months to 5 years (average 1 year). Complaints of giving-way, effusion and joint locking resolved in all patients, and pain as assessed by the visual analogue score was reduced by an average of 50%. Follow-up magnetic resonance imaging studies in all patients revealed that the defects were filled with tissue having similar signal characteristics to cartilage. CONCLUSIONS: Chondrocyte implantation is a procedure capable of restoring normal articular cartilage in cases with isolated joint defects. Pain can be predictably reduced, while joint locking and effusion are eliminated. The effect on osteoarthritis progression in humans has not yet been elucidated.

Osteogenic growth peptide normally stimulated by blood loss and marrow ablation has local and systemic effects on fracture healing in rats.

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.

Reduced NO accumulation in arthrotic cartilage by exposure to methylene blue.

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.

Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3(G380R) transgenic mice.

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.

Mesenchymal cells and growth factors in bunions.

Bunion formation in adults is an example of bone growth that occurs after physis closure. Bone is laid down secondary to mechanical irritation caused by foot deformity. It is a mechanism of ectopic bone formation unrelated to physeal growth. In this article, bone formation is analyzed using immunohistochemical and cell culture techniques. Using markers specific for mesenchymal cells (collagen type IIa and fibroblast growth factor receptor 3), a cell population is defined in the soft tissues that overlie the bunion and is isolated from explant cultures. The cells do not produce bone matrix in culture, and they do not express osteoblast-related antigens. Stimulation of the cells by fibroblast growth factor (FGF) 2 leads to rapid cell proliferation and phenotype change. The cells start to form humps and at the same time express alkaline phosphatase and collagen type I. Expression of collagen type IIa and fibroblast growth factor receptor 3 ceases. These series of experiments indicate that a specific population of mesenchymal cells occurs in the soft tissues that overlie the bunion. This population is capable of bone formation when stimulated by FGF, a common mediator of inflammatory processes. Thus, FGF stimulation of mesenchymal cells in soft tissues that overlie the head of the first metatarsal is a potential link between the biomechanical forces that cause hallux valgus and bunion formation.

Fibroblast growth factor receptor-3 as a marker for precartilaginous stem cells.

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.

Changes in proteoglycans of intervertebral disc in diabetic patients. A possible cause of increased back pain.

STUDY DESIGN: Characterization of the analytic profile of proteoglycans in the intervertebral discs at L4-L5 of nondiabetic (n = 5) and diabetic (n = 5) age-matched subjects. The discs used were discarded material from operations. OBJECTIVES: To clarify the reason for the higher risk of disc prolapse in diabetic patients. SUMMARY OF BACKGROUND DATA: The pathogenesis of diabetes results from a combination of neurologic dysfunctions and a yet undefined metabolic failure, which leads to an abnormal proteoglycan profile. METHODS: The following methods were used to determine the proteoglycan profile: the measurement of 35S-sulfate uptake per gram wet tissue into sulfated glycosaminoglycan using fresh tissue explants; extraction of proteoglycans by 4 M guanidinium chloride containing protease inhibitors, with further purification by ultracentrifugation on cesium chloride buoyant density gradient under dissociative conditions; total uronic acid and protein contents in the various gradient fractions; assessing the length of sugar side chains of isolated 35Sulfate-glycosaminoglycan molecules by separation of the glycosaminoglycan molecules on a Sepharose 6B-CL column; and paper chromatography of the final digest products of glycosaminoglycan molecules obtained by chondroitinase ABC, a glycosaminoglycan-degrading enzyme. RESULTS: The findings show that discs from normal nondiabetic subjects have 15 times the rate of 35Sulfate incorporation into glycosaminoglycan molecules than do discs of diabetic patients. The proteoglycans of diabetic patients are banded at a lower buoyant density, indicating a lowered glycosylation rate and a lower number of sugar side chains per core protein. In discs of diabetic patients, there is a slight increase in the chain length of chondroitin sulfate. Further analysis of the glycosaminoglycan chains showed a decreased amount of keratan sulfate, compared with that in nondiabetic subjects. However, the total uronic acid content of the disc tissues and the ratio of uronic acid to protein of each fraction were unchanged in diabetic patients versus that in control subjects. CONCLUSIONS: Discs in patients with diabetes have proteoglycans with lower buoyant density and substantially undersulfated glycosaminoglycan, which with the specific neurologic damage in these patients, might lead to increased susceptibility to disc prolapse.

The manipulated mesenchymal stem cells in regenerated skeletal tissues.

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.

Treatment of staphylococcal septic arthritis in rabbits by systemic antibiotics and intra-articular corticosteroids.

OBJECTIVE: To assess the effect of intra-articular corticosteroids added to systemic antibiotics in experimental septic arthritis. METHODS: Rabbits were injected intra-articularly by Staphylococcus epidermidis. Rabbits received no additional treatment and served as control (group 1), were treated with systemic antibiotics (group 2), or treated with systemic antibiotics and intra-articular corticosteroids (group 3). After 15 days animals were killed and joint histopathological-histochemical parameters were assessed. RESULTS: All rabbits survived the experiment. The treated groups (2-3) had lower histological-histochemical scores in comparison with the untreated group (1). Group 3 had significantly lower scores in joint sections in comparison with group 2: (mean (SD) 6.5 (1.4) v 4.0 (1.0), p = 0.001 and 7.4 (2.6) v 4.2 (2.2), p = 0.01), because of lower damage expressed in clustering of chondrocytes, pannus formation, proteoglycan depletion, and synovitis. CONCLUSION: Addition of local corticosteroids to systemic antibiotics in septic arthritis seems to be harmless, and improves joint histological-histochemical parameters in this experimental setting.

Does glucose affect fertilization, development and pregnancy rates of human in-vitro fertilized oocytes?

The study was conducted to examine whether the presence of glucose in the incubation medium affects fertilization, development and implantation rates of human oocytes of patients who were attending our in-vitro fertilization programme. Harvested oocytes were transferred into one of four different media: human tubal fluid (HTF), P1, M3 and IVF-Universal (IVF-Med). Three of these contained glucose; the fourth (P1), contained no glucose or phosphate ions. In an independent preliminary study, some of the oocytes of each patient were incubated in IVF-Med, which lacks phosphate ions, but not glucose. Comparisons of fertilization rates between media pairs showed differences among all pairs except HTF and M3. When comparing the four study groups, no difference was noticed in embryo development or embryo quality 48 h post-ovum retrieval. A higher development rate was demonstrated in embryos incubated in M3 medium, in comparison with the P1 and IVF-Med embryos after incubation for 72 h. No difference in pregnancy rate was found after embryo transfers of preimplantation embryos which were incubated in one of the following media: HTF, M3 and IVF-Med (seven out of 22, 18 of 54 and 32 of 69 treatment cycles respectively). A lower incidence of pregnancies occurred following transfers of embryos which were incubated in P1 medium (seven pregnancies out of 37 cycles). We suggest that the presence of glucose in the incubation medium enhances implantation potential of in-vitro-developing preimplantation embryos.

The radiographic stage of giant cell tumor related to stromal cells' proliferation. Tissue cultures in 13 cases.

The clinical behavior of giant cell tumor is related to the radiological appearance. To test the hypothesis that in vitro proliferation of the neoplastic stromal cell population of giant cell tumors is related to the radiological appearance, this study was undertaken. A prospective analysis of the cells migrating from 13 consecutive tumors was conducted. Growth curves and population doubling-times (PDT) for first and fifth passages were calculated and alkaline phosphatase levels were measured and compared to preoperative radiographic staging. A strong negative correlation was found between PDT and the radiographic stage. Tumors in stages I and II (low aggressiveness) were found to have an average cell population doubling-time of 11 (SD 2.2) days, while those in stage III (high aggressiveness) showed a doubling-time of 6 (SD 2.2) days. Low alkaline phosphatase activity was noted in all cultures, a finding consistent with the putative preosteoblastic potential of these stromal cells. This putative origin is also indicated by the differentiation response to retinoic acid. The findings suggest that the in vitro proliferation of the mononuclear stromal cell population of giant cell tumors is related to the radiographic stage and may predict the clinical behavior of these tumors.