Zvi Nevo

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.

Hyaluronan and mesenchymal stem cells: from germ layer to cartilage and bone.

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.

Increase of neuronal sprouting and migration using 780 nm laser phototherapy as procedure for cell therapy.

The present study focuses on the effect of 780 nm laser irradiation on the growth of embryonic rat brain cultures embedded in NVR‐Gel (cross‐linked hyaluronic acid with adhesive molecule laminin and several growth factors). Dissociated neuronal cells were first grown in suspension attached to cylindrical microcarriers (MCs). The formed floating cell‐MC aggregates were subsequently transferred into stationary cultures in gel and then laser treated. The response of neuronal growth following laser irradiation was investigated.

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.

Transplantation of embryonal spinal cord nerve cells cultured on biodegradable microcarriers followed by low power laser irradiation for the treatment of traumatic paraplegia in rats

Abstract 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 control group 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. [Neurol Res 2002; 24: 355-360]

Slowing down aging of cultured embryonal chick chondrocytes by maintenance under lowered oxygen tension.

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 [Ca2+]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 [Ca2+]in in these cells.