Adult mesenchymal stem cells (MSCs) have the capacity to differentiate into various connective tissues such as cartilage and bone following stimulation with certain growth factors. However, less is known about the capacity of these cells to undergo chondrogenesis when these proteins are delivered via gene transfer. In this study, we investigated chondrogenesis of primary, bone marrow-derived MSCs in aggregate cultures following genetic modification with adenoviral vectors encoding chondrogenic growth factors. We found that adenoviral-mediated expression of TGF-beta1 and BMP-2, but not IGF-1, induced chondrogenesis of MSCs as evidenced by toluidine blue metachromasia and immunohistochemical detection of type II collagen. Chondrogenesis correlated with the level and duration of expressed protein and was strongest in aggregates expressing 10-100 ng/ml transgene product. Transgene expression in all aggregates was highly transient, showing a marked decrease after 7 days. Chondrogenesis was inhibited in aggregates modified to express >100 ng/ml TGF-beta1 or BMP-2; however, this was found to be partly due to the inhibitory effect of exposure to high adenoviral loads. Our findings indicate that parameters such as these are important functional considerations for adapting gene transfer technologies to induce chondrogenesis of MSCs.

The long-term goal of the present study is to develop a clinically applicable approach to enhance natural repair mechanisms within cartilage lesions by targeting bone marrow-derived cells for genetic modification. To determine if bone marrow-derived cells infiltrating osteochondral defects could be transduced in situ, we implanted collagen-glycosaminoglycan (CG) matrices preloaded with adenoviral vectors containing various marker genes into lesions surgically generated in rabbit femoral condyles. Analysis of the recovered implants showed transgenic expression up to 21 days; however, a considerable portion was found in the synovial lining, indicating leakage of the vector and/or transduced cells from the matrix. As an alternative medium for gene delivery, we investigated the feasibility of using coagulated bone marrow aspirates. Mixture of an adenoviral suspension with the fluid phase of freshly aspirated bone marrow resulted in uniform dispersion of the vector throughout, and levels of transgenic expression in direct proportion to the density of nucleated cells in the ensuing clot. Furthermore, cultures of mesenchymal progenitor cells, previously transduced ex vivo with recombinant adenovirus, were readily incorporated into the coagulate when mixed with fresh aspirate. These vector-seeded and cell-seeded bone marrow clots were found to maintain their structural integrity following extensive culture and maintained transgenic expression in this manner for several weeks. When used in place of the CG matrix as a gene delivery vehicle in vivo, genetically modified bone marrow clots were able to generate similarly high levels of transgenic expression in osteochondral defects with better containment of the vector within the defect. Our results suggest that coagulates formed from aspirated bone marrow may be useful as a means of gene delivery to cartilage and perhaps other musculoskeletal tissues. Cells within the fluid can be readily modified with an adenoviral vector, and the matrix formed from the clot is completely natural, native to the host and is the fundamental platform on which healing and repair of mesenchymal tissues is based.

Facilitated endogenous repair is a novel approach to tissue engineering that avoids the ex vivo culture of autologous cells and the need for manufactured scaffolds, while minimizing the number and invasiveness of associated clinical procedures. The strategy relies on harnessing the intrinsic regenerative potential of endogenous tissues using molecular stimuli, such as gene transfer, to initiate reparative processes in situ. In the simplest example, direct percutaneous injection of an osteogenic vector is used to stimulate bone healing. If necessary, additional progenitor cells and space-filling scaffolds can be provided by autologous bone marrow, muscle, fat, and perhaps other tissues. These can be harvested, processed, and reimplanted by simple, expedited, intraoperative procedures. Examples of repair of experimental osseous and osteochondral lesions in laboratory animals are described. If successful, these strategies will provide methods for tissue regeneration that are not only effective but also inexpensive, safe, and clinically expeditious. Although orthopaedic examples are given here, the technology should be more generally applicable.

No abstract

The inability of the ruptured anterior cruciate ligament (ACL) of the knee joint to heal spontaneously presents numerous clinical problems. Here we describe a novel, gene-based approach to augment ACL healing. It is based upon the migration of cells from the ruptured ends of the ligament into a collagen hydrogel laden with recombinant adenovirus. Cells entering the gel become transduced by the vector, which provides a basis for the local synthesis of gene products that aid repair. Monolayers of bovine ACL cells were readily transduced by first-generation, recombinant adenovirus, and transgene expression remained high after the cells were incorporated into collagen hydrogels. Using an in vitro model of ligament repair, cells migrated from the cut ends of the ACL into the hydrogel and were readily transduced by recombinant adenovirus contained within it. The results of experiments in which GFP was used as the transgene suggest highly efficient transduction of ACL cells in this manner. Moreover, during a 21-day period GFP+ cells were observed more than 6 mm from the severed ligament. This distance is ample for the projected clinical application of this technology. In response to TGF-beta1 as the transgene, greater numbers of ACL cells accumulated in the hydrogels, where they deposited larger amounts of type III collagen. These data confirm that it is possible to transduce ACL cells efficiently in situ as they migrate from the ruptured ACL, that transduction does not interfere with the cells' ability to migrate distances necessary for successful repair, and that ACL cells will respond in a suitable manner to the products of the transgenes they express. This permits optimism over a possible clinical use for this technology.

We performed a retrospective analysis of the clinical and radiological outcomes of total hip replacement using an uncemented femoral component proximally coated with hydroxyapatite. Of 136 patients, 118 who had undergone 124 primary total hip replacements were available for study. Their mean age was 66.5 years (19 to 90) and the mean follow-up was 5.6 years (4.25 to 7.25). At the final follow-up the mean Harris hip score was 92 (47.7 to 100). Periprosthetic femoral fractures, which occurred in seven patients (5.6%), were treated by osteosynthesis in six and conservatively in one. We had to revise five femoral components, one because of aseptic loosening, one because of septic loosening and three because of periprosthetic fracture. At the final follow-up there were definite signs of aseptic loosening in two patients. Radiologically, proximal femoral bone loss in Gruen zones I and VI was evident in 96.8% of hips, while bone hypertrophy in zones III and V was seen in 64.7%. In 24 hips (20.2%) the mean subsidence of the stem was 3.7 mm which occurred within the first 12 postoperative weeks. This indicated poor initial stability, which might have been aggravated by early weight-bearing. The high rate of failure in our study suggests that proximal femoral bone loss affects the long-term survival of the replacement.

No abstract

We performed a retrospective analysis of the clinical and radiological outcomes of total hip replacement using an uncemented femoral component proximally coated with hydroxyapatite. Of 136 patients, 118 who had undergone 124 primary total hip replacements were available for study. Their mean age was 66.5 years (19 to 90) and the mean follow-up was 5.6 years (4.25 to 7.25). At the final follow-up the mean Harris hip score was 92 (47.7 to 100). Periprosthetic femoral fractures, which occurred in seven patients (5.6%), were treated by osteosynthesis in six and conservatively in one. We had to revise five femoral components, one because of aseptic loosening, one because of septic loosening and three because of periprosthetic fracture. At the final follow-up there were definite signs of aseptic loosening in two patients.Radiologically, proximal femoral bone loss in Gruen zones I and VI was evident in 96.8% of hips, while bone hypertrophy in zones III and V was seen in 64.7%. In 24 hips (20.2%) the mean subsidence of the stem was 3.7 mm which occurred within the first 12 postoperative weeks. This indicated poor initial stability, which might have been aggravated by early weight-bearing. The high rate of failure in our study suggests that proximal femoral bone loss affects the long-term survival of the replacement.