Brian Johnstone

The Chondrogenic Potential of Human Bone-marrow-derived Mesenchymal Progenitor Cells*

Mesenchymal progenitor cells provide a source of cells for the repair of musculoskeletal tissue. However, in vitro models are needed to study the mechanisms of differentiation of progenitor cells. This study demonstrated the successful induction of in vitro chondrogenesis with human bone-marrow-derived osteochondral progenitor cells in a reliable and reproducible culture system. Human bone marrow was removed and fractionated, and adherent cell cultures were established. The cells were then passaged into an aggregate culture system in a serum-free medium. Initially, the cell aggregates contained type-I collagen and neither type-II nor type-X collagen was detected. Type-II collagen was typically detected in the matrix by the fifth day, with the immunoreactivity localized in the region of metachromatic staining. By the fourteenth day, type-II and type-X collagen were detected throughout the cell aggregates, except for an outer region of flattened, perichondrial-like cells in a matrix rich in type-I collagen. Aggrecan and link protein were detected in extracts of the cell aggregates, providing evidence that large aggregating proteoglycans of the type found in cartilaginous tissues had been synthesized by the newly differentiating chondrocytic cells; the small proteoglycans, biglycan and decorin, were also detected in extracts. Immunohistochemical staining with antibodies specific for chondroitin 4-sulfate and keratan sulfate demonstrated a uniform distribution of proteoglycans throughout the extracellular matrix of the cell aggregates. When the bone-marrow-derived cell preparations were passaged in monolayer culture as many as twenty times, with cells allowed to grow to confluence at each passage, the chondrogenic potential of the cells was maintained after each passage. CLINICAL RELEVANCE: Chondrogenesis of progenitor cells is the foundation for the in vivo repair of fractures and damaged articular cartilage. In vitro chondrogenesis of human bone-marrow-derived osteochondral progenitor cells should provide a useful model for studying this cellular differentiation. Furthermore, the maintenance of chondrogenic potential after greater than a billion-fold expansion provides evidence for the clinical utility of these cells in the repair of bone and cartilage.

A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse.

Adult marrow contains mesenchymal progenitor cells (MPCs) that have multiple differentiation potentials. A conditionally immortalized MPC clone, BMC9, has been identified that exhibits four mesenchymal cell phenotypes: chondrocyte, adipocyte, stromal (support osteoclast formation), and osteoblast. The BMC9 clone, control brain fibroblasts and another marrow‐derived clone, BMC10, were isolated from a transgenic mouse (H‐2Kb‐tsA58) containing a gene for conditional immortality. To test for chondrogenic potential, cells were cultured in defined medium containing 10 ng/ml transforming growth factor β and 10−7 M dexamethasone in 15‐ml polypropylene tubes (“aggregate cultures”). Adipogenic potential was quantitated by flow cytometry of Nile Red–stained cells cultured for 1 and 2 weeks in medium containing isobutyl methylxanthine, indomethacin, insulin, and dexamethasone. Support of osteoclast formation was measured by quantitating multinucleated tartrate‐resistant acid phosphatase–positive cells in spleen cell cocultures of test clones (immortomouse clones and positive control ST2 cells) cultured in the presence of 10−7 M vitamin D3 and 150 mM ascorbate‐2‐phosphate. In vivo osteogenic potential was assayed by histologic examination of bone formation in subcutaneous implants, into athymic mouse hosts, of a composite of cells combined with porous calcium phosphate ceramics. The bone marrow–derived clone BMC9 has the potential to express each of the four mesenchymal characteristics tested, while brain fibroblasts, tested under identical conditions, did not exhibit any of these four mesenchymal characteristics. BMC10 cells exhibited osteogenic and chondrogenic phenotypes, but showed only minimal expression of adipocytic or osteoclast‐supportive phenotypes. Clone BMC9 is, minimally, a quadripotential MPC isolated from the marrow of an adult mouse that can differentiate into cartilage and adipose, support osteoclast formation, and form bone. The BMC9 clone is an example of an adult‐derived multipotential progenitor cell that is situated early in the mesenchymal lineage.

Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro

Much attention has been given to the influences of bioactive factors on mesenchymal progenitor cell differentiation and proliferation, but few studies have examined the effect of mechanical factors on these cells. This study examined the effects of cyclic hydrostatic pressure on human bone marrow‐derived mesenchymal progenitor cells undergoing chondrogenic differentiation. Aggregates of bone marrow‐derived mesenchymal progenitor cells were cultured in a defined chondrogenic medium and were subjected to cyclic hydrostatic pressure. Aggregates were loaded at various time points: single (day 1 or 3) or multiple (days 1–7). At 14 and 28 days, aggregates were harvested for histology, immunohistochemistry, and quantitative DNA and matrix macromolecule analysis. The aggregates loaded for a single day did not demonstrate significant changes in proteoglycan and collagen contents compared with the non‐loaded controls. In contrast, for the multi‐day loaded aggregates, statistically significant increases in proteoglycan and collagen contents were found on both day 14 and day 28. Aggregates loaded for seven days were larger and histological staining indicated a greater matrix/cell ratio. This study indicates that hydrostatic pressure enhances the cartilaginous matrix formation of mesenchymal progenitor cells differentiated in vitro, and suggests that mechanical forces may play an important role in cartilage repair and regeneration in vivo.

Effect of IGF‐I in the Chondrogenesis of Bone Marrow Mesenchymal Stem Cells in the Presence or Absence of TGF‐β Signaling

A novel role for IGF‐I in MSC chondrogenesis was determined. IGF‐I effects were evaluated in the presence or absence of TGF‐β signaling by conditionally inactivating the TGF‐β type II receptor. We found that IGF‐I had potent chondroinductive actions on MSCs. IGF‐I effects were independent from and additive to TGF‐β.

CHONDROPROGENITOR CELLS OF SYNOVIAL TISSUE

OBJECTIVE To assess the chondrogenic potential of cells within the synovium. METHODS Explants of synovium taken from various sites in the joint were embedded in agarose and cultured with transforming growth factor beta1 (TGFbeta1) to assess their chondrogenic potential. Isolated synovial cells were also tested for their chondrogenic potential by culturing them as aggregates in a chemically defined medium with TGFbeta1. Cartilage formation was determined with histologic staining and immunohistochemistry. The osteochondral potential of the isolated cells was also assessed after subcutaneous implantation of the cells, loaded into porous calcium phosphate ceramic cubes, in athymic mice. RESULTS A total of 48 synovial explants were cultured in agarose with TGFbeta1. The formation of cartilage was observed in the outer region of 21 explants, and type II collagen was localized in that region by immunohistochemistry. A larger percentage of TGFbeta1+ explants from the inner synovium sites formed cartilage compared with those from the outer synovium sites. Chondrogenesis occurred in aggregates incubated with TGFbeta1 as early as day 7, and by day 14, all TGFbeta1+ aggregates demonstrated chondrogenesis. In contrast with the results of the in vitro aggregate assay for chondrogenesis, no formation of cartilage or bone was evident in any section containing synovial cell-loaded ceramic cubes that were harvested at either 3 or 6 weeks after implantation subcutaneously in athymic mice. CONCLUSION Synovial explants and isolated synovial cells will undergo chondrogenesis when cultured in the presence of TGFbeta1. The data indicate a possible synovial origin for the chondrocytic cells found in rheumatoid pannus. Furthermore, these data are consistent with the clinical findings of synovial chondrogenesis leading to synovial chondromatosis.

BMP‐2 induction and TGF‐β1 modulation of rat periosteal cell chondrogenesis

Periosteum contains osteochondral progenitor cells that can differentiate into osteoblasts and chondrocytes during normal bone growth and fracture healing. TGF‐β1 and BMP‐2 have been implicated in the regulation of the chondrogenic differentiation of these cells, but their roles are not fully defined. This study was undertaken to investigate the chondrogenic effects of TGF‐β1 and BMP‐2 on rat periosteum‐derived cells during in vitro chondrogenesis in a three‐dimensional aggregate culture. RT‐PCR analyses for gene expression of cartilage‐specific matrix proteins revealed that treatment with BMP‐2 alone and combined treatment with TGF‐β1 and BMP‐2 induced time‐dependent mRNA expression of aggrecan core protein and type II collagen. At later times in culture, the aggregates treated with BMP‐2 exhibited expression of type X collagen and osteocalcin mRNA, which are markers of chondrocyte hypertrophy. Aggregates incubated with both TGF‐β1 and BMP‐2 showed no such expression. Treatment with TGF‐β1 alone did not lead to the expression of type II or X collagen mRNA, indicating that this factor itself did not independently induce chondrogenesis in rat periosteal cells. These data were consistent with histological and immunohistochemical results. After 14 days in culture, BMP‐2‐treated aggregates consisted of many hypertrophic chondrocytes within a metachromatic matrix, which was immunoreactive with anti‐type II and type X collagen antibodies. In contrast, at 14 days, TGF‐β1+BMP‐2‐treated aggregates did not contain any morphologically identifiable hypertrophic chondrocytes and their abundant extracellular matrix was not immunoreactive to the anti‐type X collagen antibody. Expression of BMPR‐IA, TGF‐β RI, and TGF‐β RII receptors was detected at all times in each culture condition, indicating that the distinct responses of aggregates to BMP‐2, TGF‐β1 and TGF‐β1+BMP‐2 were not due to overt differences in receptor expression. Collectively, our results suggest that BMP‐2 induces neochondrogenesis of rat periosteum‐derived cells, and that TGF‐β1 modulates the terminal differentiation in BMP‐2 induced chondrogenesis. J. Cell. Biochem. 80:284–294, 2001.

Genetic enhancement of matrix synthesis by articular chondrocytes: Comparison of different growth factor genes in the presence and absence of interleukin‐1

OBJECTIVE To determine whether articular chondrocytes express growth factor genes delivered by adenoviral vectors and whether expression of these genes influences matrix synthesis in the presence and absence of interleukin-1 (IL-1). METHODS Monolayer cultures of rabbit articular chondrocytes were infected with recombinant adenovirus carrying genes encoding the following growth factors: insulin-like growth factor 1 (IGF-1), transforming growth factor beta1 (TGFbeta1), and bone morphogenetic protein 2 (BMP-2). As a control, cells were transduced with the lac Z gene. Cultures were also treated with each growth factor supplied as a protein. Levels of gene expression were noted, and the synthesis of proteoglycan, collagen, and noncollagenous proteins was measured by radiolabeling. Collagen was typed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The effects of growth factor gene transfer on proteoglycan synthesis in the presence of IL-1 were also measured. RESULTS The expression of all transgenes was high following adenoviral transduction. Proteoglycan synthesis was stimulated approximately 8-fold by the BMP-2 gene and 2-3-fold by the IGF-1 gene. The effects of BMP-2 and IGF-1 genes were additive upon cotransduction. Synthesis of collagen and noncollagenous proteins, in contrast, was most strongly stimulated by the IGF-1 gene. In each case, collagen typing confirmed the synthesis of type II collagen. IL-1 suppressed proteoglycan synthesis by 50-60%. IGF-1 and TGFbeta genes restored proteoglycan synthesis to control levels in the presence of IL-1. The BMP-2 gene, in contrast, elevated proteoglycan synthesis beyond control levels in the presence of IL-1. CONCLUSION Transfer of growth factor genes to articular chondrocytes can greatly increase matrix synthesis in vitro, even in the presence of the inflammatory cytokine IL-1. This result encourages the further development of gene therapy for the repair of damaged cartilage.

Autologous mesenchymal progenitor cells in articular cartilage repair.

The lack of repair of articular cartilage where the damage does not penetrate the subchondral bone indicates the importance of marrow components in the repair of the articular cartilage. In adult animals, there is an inability of articular cartilage chondrocytes to heal chondral defects, but if the damage extends beyond the subchondral bone, a repair process ensues in which mesenchymal progenitor cells migrate into the injured site and undergo chondrogenic differentiation. However, analysis of animal models and human biopsy samples indicates that fibrocartilage, rather than true articular cartilage is the predominant tissue synthesized. To improve this outcome, the use of cell based implants of culture expanded progenitor cells from various sources has been proposed and attempted. This paper describes some of the age related differences in the natural repair of osteochondral defects, the in vitro characterization of the chondrogenic potential of certain mesenchymal cell types, and some of the characteristics required of cell and matrix constructs that may be used for repair or regeneration of articular cartilage.

Morphologic considerations of C2 isthmus dimensions for the placement of transarticular screws.

Study Design. This study examines the C2 vertebrae using both direct anatomic and computed tomographic measurements. Objective. To define the relation of the C2 vertebrae bony elements to the vertebral artery and the spinal canal, to determine individuals at risk for vertebral artery injury during C1–C2 transarticular screw placement. Summary of Background Data. Recent literature assessing the safety of upper cervical spine transarticular screws has concentrated on technique, including the optimal point of entry and path projection of the screw. The actual dimensions of the C2 isthmus of the pars interarticularis has not been examined in a large number of specimens. Methods. C2 isthmus width and height measurements were made on 205 human cadaveric C2 vertebrae. Vertebrae predicted to be at risk for vertebral arterial injury were imaged by computed tomography. Results. There were 102 female and 103 male specimens with mean isthmus widths of 8.2 ± 1.5 mm and 7.2 ± 1.3 mm, respectively. Five specimens (2.4%) had an isthmus width less than 5 mm. The mean isthmus heights were 8.6 ± 2.0 mm and 6.9 ± 1.5 mm for male and female specimens, respectively. In twenty-four specimens (11.7%), one or both isthmi had a height of less than 5 mm. Six of these specimens were male and 18 were female. The right C2 isthmus was generally smaller than the left. Computed tomographic measurements closely approximated those of the actual dimensions of the isthmi. Conclusions. Placing a 3.5 mm screw in a patient with narrow C2 isthmus dimensions (smaller than 5 mm in either the height or width) is technically difficult. Because of narrow C2 isthmus width and/or height, approximately 10% of patients may be at risk for a vertebral artery injury with placement of C1–C2 transarticular screws.

The large proteoglycans of the human intervertebral disc. Changes in their biosynthesis and structure with age, topography, and pathology.

Study Design The structure and extracellular assembly of the newly sythesized aggregating proteoglycans of the human intervertebral disc were examined using an explant culture system. Objectives The objective was to study the changes with aging, topography, and pathology, comparing newly synthesized with endogenous proteoglycans. Summary of Background Data No detailed studies of the biosynthesis of human disc proteglycans have been previously reported. Methods A method of explant culture that minimizes swelling and matrix loss was used to maintain the tissue architecture. Slices of postmortem and pathologic disc tissues were incubated in medium containing polyethylene glycol at appropriate concentrations to balance the swelling pressure of the tissue. The disc slices were contained in small-pore dialysis tubing to prevent penetration of the polyethylene glycol into the tissue. The newly synthesized proteoglycans were radiolabled with [35S]-sulphate. Proteoglycans were then extracted from the tissue slices and characterized with gel chromatographic and electrophoretic techniques. Results It was found that a single, high molecular weight proteoglycan is the major 35S-labeled synthesis product of disc cells at all ages. However, biosynthetic changes do occur: the monomer made by fetal and newborn disc cells was larger than that of adults. Furthermore, adult disc cells made other minor large 35S-labeled products, the synthesis pattern of which varied between regions. Conclusion These results provide the first evidence that biosynthetic changes contribute to the age-related increase in the heterogeneity of the human disc proteoglycan population.