Dror Robinson

Resurfacing of goat articular cartilage by chondrocytes derived from bone marrow.

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.

Regenerating hyaline cartilage in articular defects of old chickens using implants of embryonal chick chondrocytes embedded in a new natural delivery substance

SummaryPartial and full thickness defects were created mechanically in articular cartilage and subchondral bone of the tibiotarsal joint condyles of 3-year-old chickens. The wounds were then repaired using embryonal chick chondrocytes embedded in a new biocompatible, hyaluronic acid-based delivery substance. Controls were similarly operated on but received either no treatment or implants of the delivery substance only. Animals were killed from 1 week to 6 months postoperatively. Sections from the two groups were examined and compared macroscopically, histologically, and histochemically. Results of 6-month follow-up showed that only the defects of the experimental chickens were completely filled with reparative hyaline cartilage tissue, with no signs of inflammation or immunologic rejection. Initially the entire defect cavity, whether partial thickness or full thickness up to the deep regions in the subchondral bone, was filled with cartilaginous reparative tissue. Relatively rapid maturation occurred under the tidemark; chondrocytes hypertrophied, were invaded with vascular elements and ossified. In the superficial areas, the reparative tissue remained cartilaginous and matured as typical hyaline cartilage tissue. These results indicate that aged chicken cartilage and its accompanying thin and spongy osteoporotic bone offer a favorable host environment for embryonal cell implants.

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 35 Sulfate‐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.

Anterior compartment syndrome of the thigh in athletes-indications for conservative treatment

Anterior compartment syndrome of the thigh resulting from blunt contusion without an accompanying fracture is rare. The treatment advocated for it by most authors has been surgical. However, because wound infection rates are high and loss of knee range of motion is frequent, we considered conservative treatment in selected patients. Six athletes who developed an anterior thigh compartment syndrome shortly after sustaining a blunt contusion to the quadriceps with an accompanying massive hematoma were studied prospectively. Tissue pressure, renal function, and creatinine phosphokinase (CPK) levels were closely monitored. Fasciotomy was not performed, despite sustained pressure elevations above 50 mm Hg. Neurologic function was not affected. At follow-up examination 1 year later, no limitation of joint motion nor weakness of the quadriceps were observed. Thus in selected young patients in whom an isolated anterior compartment syndrome of the thigh occurs, conservative treatment yielded results superior to fasciotomy.

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.

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.

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.

Shoulder girdle neoplasms mimicking frozen shoulder syndrome

Shoulder pain and immobility comprise a multifactorial disorder apparently affected by pain inhibiting joint motion. As the syndrome is very common, many patients do not undergo detailed imaging studies before treatment. This study compared a series of 7 patients in whom a neoplasm was the underlying cause for the stiff shoulder with a series of 50 patients with primary or secondary frozen shoulder. In addition to a detailed history being taken, the Disabilities of the Arm, Shoulder, and Hand (DASH) upper limb outcomes data collection questionnaire was completed and physical examination, radiography, ultrasonography, and bone scanning were performed in all cases. In the cases of tumor, the presenting symptom was a stiff shoulder without radiographic abnormality in 7 of 67 patients with shoulder girdle neoplasms who were seen at our musculoskeletal oncology clinic. The tumors included osteoid osteoma, osteoblastoma, metastatic carcinoma, chondrosarcoma, periosteal lipoma, and acute lymphoblastic lymphoma. The diagnosis was established in all cases by an area of focal isotope uptake demonstrated by a routine technetium 99 methylene diphosphonate bone scan. In a single case of metastatic colon carcinoma, the diagnosis could only be established by magnetic resonance imaging, as the radiographs were normal and the bone scan demonstrated diffuse uptake over the proximal humerus. The patients whose frozen shoulder was caused by an underlying tumor were significantly younger and had a lower fatigue/energy dimension score on the RAND Short Form-36 health survey. The most useful diagnostic test appears to be a discrete area of bony tenderness, present in 7 of 7 patients with tumor and in only 5 of 50 patients in the control group. Although an underlying tumor is a rare cause of frozen shoulder syndrome, the potential grave consequences of misdiagnosis and the possibility of performing an unnecessary and ineffective invasive procedure should prompt physicians to increased vigilance. In patients with discrete bony tenderness elicited by light tapping, a bone scan should be ordered and magnetic resonance imaging should be considered.

Hyperelastic mechanical behavior of chitosan hydrogels for nucleus pulposus replacement—Experimental testing and constitutive modeling

Chitosan hydrogels (CHs) have been considered as a potential implant material for replacement and repair of the Nucleus Pulposus (NP) within the intervertebral disk. The nonlinear mechanical behavior of a CH material is investigated experimentally and computationally in this study. A series of confined and unconfined compression tests are designed and conducted for this hydrogel. Hyperelastic strain energy density functions (SEDFs) are calibrated using the experimental data. A hyperelastic constitutive model is selected to best fit the multi-axial behavior of the hydrogel. Its general prediction ability is verified using finite element (FE) simulations of hydrogel indentation experiments conducted using a spherical tip indentor. In addition, digital image correlation (DIC) technique is also used in the indentation test in order to process the full-field surface strains where the indentor contacts the hydrogel. The DIC test results in the form of top-surface strains compared well with those predicted by the FE model. Results show repeatability for the examined specimens under the applied tests. Confined and unconfined test results are found to be sufficient to calibrate the SEDFs. The Ogden model was selected to represent the nonlinear behavior of the CH material which can be used in future biomechanical simulations of the spine.

The molecular and cellular basis of exostosis formation in hereditary multiple exostoses

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