Jung U. Yoo

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.

Incidence of dysphagia after anterior cervical spine surgery: a prospective study.

Study Design. A prospective longitudinal study was conducted to evaluate dysphagia after anterior cervical spine surgery. Objectives. To evaluate the incidence and natural history of dysphagia after anterior cervical spine surgery, and to identify risk factors for the development of postoperative dysphagia. Summary of Background Data. The literature contains only retrospective evaluations of postoperative dysphagia. A wide range of incidence has been reported in these studies. Methods. Altogether, 249 consecutive patients undergoing anterior cervical spine surgery were eligible for the study. These patients were contacted 1, 2, 6, and 12 months after the procedure to evaluate swallowing. Risk factors such as age, gender, procedure type, hardware use, and number and location of surgical levels addressed were assessed. Results. Dysphagia incidences of 50.2%, 32.2%, 17.8%, and 12.5% were found at 1, 2, 6, and 12 months, respectively. At 6 months after the procedure, only 4.8% of the patients were experiencing moderate or severe dysphagia. Patient age, type of procedure (corpectomy vs. discectomy or primary vs. revision), hardware presence, and location of surgical levels were not statistically significant risk factors for the development of postoperative dysphagia. Female gender was significant for increased risk of dysphagia at 6 months. Surgery at multiple disc levels increased the risk of postoperative dysphagia at 1 and 2 months. The etiology of the dysphagia in most of the patients was unknown. However, vocal cord paresis was identified in 1.3% of the patients at 12 months. Conclusions. Dysphagia after anterior cervical spine surgery is a common early finding. However, it decreases significantly by 6 months. The minority of patients experience moderate or severe symptoms by 6 months after the procedure. Female gender and multiple surgical levels could be identified as risk factors for the development of postoperative dysphagia.

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.

The success of anterior cervical arthrodesis adjacent to a previous fusion.

Study Design. A retrospective review of all patients surgically treated for adjacent segment disease of the cervical spine over a 20‐year period. Objectives. To determine the clinical and radiographic success of discectomy with interbody grafting and corpectomy with strut grafting in the treatment of adjacent segment disease of the cervical spine. Summary of Background Data. Up to 25% of all patients undergoing anterior cervical fusion have new disease due to degeneration of an adjacent segment within 10 years. The success of surgical treatment in these patients with adjacent segment disease has not been reported. Methods. Thirty‐eight patients were surgically treated for adjacent segment disease by discectomy with interbody grafting or corpectomy with strut grafting. Arthrodesis was evaluated by flexion‐extension lateral radiographs and clinical outcomes were assessed using Robinsons criteria at least 2 years after surgery. Fusion rates were compared by Fishers exact test, and outcomes were compared by rank‐sum analysis. Results. The rate of arthrodesis was significantly lower in the 24 patients treated by discectomy with interbody grafting at one or more levels (63%) than in the 14 patients treated by corpectomy with strut grafting (100%; P = 0.01). Clinical outcomes were similar for the corpectomy and discectomy groups (P = 0.55). There was a trend toward better outcomes in patients who achieved a solid arthrodesis (P = 0.13). Conclusions. Achieving fusion is more difficult when anterior cervical arthrodesis is performed adjacent to a prior fusion. Strut grafting resulted in a significantly higher rate of arthrodesis than interbody grafting.

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.

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.

Influence of anterior cervical plate design on Dysphagia : a 2-year prospective longitudinal follow-up study

Objective: To compare the incidence, prevalence, and rate of improvement of dysphagia in patients undergoing anterior cervical spine surgery with two different anterior instrumentation designs. Methods: The study subjects were 156 consecutive patients undergoing anterior cervical spine surgery with plate fixation. We compared the incidence of dysphagia among the two different plate groups both produced by the same manufacturer (Medtronic Danek); the Atlantis plate has thicker and wider plate dimensions than the Zephir plate. Dysphagia evaluations were performed prospectively by telephone interviews at 1, 2, 6, 12, and 24 months following the procedure. Risk factors such as gender, revision surgery, and number of surgical levels were compared between the groups and were not statistically different. Results: Overall incidences of dysphagia were 49%, 37%, 20%, 15.4%, and 11% at 1, 2, 6, 12, and 24 months, respectively. Severe and disabling dysphagia is reported to be a relatively uncommon complication of anterior cervical surgery. However, a significant number of patients report mild to moderate discomfort including double-swallowing and catching sensation. Except at the 2-month follow-up point, the Atlantis plate group had higher incidences of dysphagia than the Zephir group at all time points (57% vs 50%, 36% vs 4%, 23% vs 14%, 17% vs 7%, 14% vs 0% at 1, 2, 6, 12, and 24 months, respectively). The Atlantis plate group had a 14% incidence of dysphagia at 2 years compared with the Zephir group, which had a 0% incidence at 2 years (P < 0.04). For primary surgeries, there was a higher incidence of dysphagia at all time points in the Atlantis group when compared with the Zephir group (58% vs 43%, 35% vs 30%, 22% vs 10%, 17% vs 0%, and 13% vs 0% at 1, 2, 6, 12, and 24 months, respectively) (P < 0.04 at 1 year). A regression analysis was performed. The resulting formulas predict the permanent rate of dysphagia for the Atlantis group is 13.6% and for the Zephir group is 3.58%. Conclusions: The use of a smaller and smoother profile plate such as the Zephir does reduce the incidence of dysphagia as compared with a slightly larger and less smooth plate such as the Atlantis.

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.