Donor cell fate in tissue engineering for articular cartilage repair.

Articular cartilage repair is a clinical challenge because of its limited intrinsic healing potential. Considerable research has focused on tissue engineering and transplantation of viable chondrogenic cells to enhance cartilage regeneration. However, the question remains: do transplanted allogenic cells survive in the repair with time? This study assessed donor cell fate after transplantation of male New Zealand White rabbit perichondrium cell and polylactic acid constructs into osteochondral defects created in the medial femoral condyles of female New Zealand White rabbits. Repair tissue was harvested at 0, 1, 2, 3, 7, and 28 days after implantation and was evaluated for cell viability and total cell number using confocal microscopic analysis. The number of donor cells in each sample was estimated using quantitative polymerase chain reaction targeting a gender-specific gene present on the Y-chromosome, the sex-determining region Y gene, and a control deoxyribonucleic acid present in male and female cell deoxyribonucleic acid, the matrix metalloproteinase-1 gene promoter. Average cell viability was found to be 87% or more at all times. Donor cells were present in repair tissue for 28 days after implantation. However, the number of donor cells declined from approximately 1 million at Time 0 to approximately 140,000 at 28 days. This decline in donor cells was accompanied by a significant influx of host cells into the repair tissue. This study shows that the sex-determining region Y gene is a valuable marker for tracking the fate of transplanted allogenic cells in tissue engineering.

The tetrabasic KKKK(147-150) motif determines intracrine regulatory effects of PthrP 1-173 on chondrocyte PPi metabolism and matrix synthesis.

Expression of PTHrP is a major regulator of growth cartilage development and also becomes robust in osteoarthritic cartilage. We further defined how PTHrP 1-173, which we observed to be the preferentially expressed PTHrP isoform in normal and osteoarthritic cartilage, functions in chondrocytes. We transfected both immortalized human juvenile costal chondrocytes (TC28 cells) and rabbit articular chondrocytes with wild-type PTHrP 1-173 and mutants of putative PTHrP 1-173 endoproteolytic processing sites. Wild-type PTHrP 1-173 inhibited collagen synthesis and decreased extracellular PPi (which critically regulates hydroxyapatite deposition) by 50-80% in both chondrocytic cell types. In contrast, PTHrP 1-173 mutated at the PTHrP 147-150 motif KKKK (but not the other site-directed mutants) and increased both extracellular PPi and collagen synthesis by >50%. Synthetic PTHrP 140-173 mutated at amino acids 147-150 and also increased extracellular PPi, and wild-type 140-173 decreased extracellular PPi in permeabilized cells. The 147-nuclear localization of PTHrP. We conclude that the tetrabasic 147-150 motif functions to determine how PTHrP 1-173 regulates collagen synthesis and levels of extracellular PPi by an intracrine mechanism in chondrocytes, and it may prove useful as a therapeutic target for regulation of mineralization.

Nonviral in vivo gene therapy for tissue engineering of articular cartilage and tendon repair.

Heretofore, nonviral methods have been used primarily for in vitro transfection of cultured cell lines. These methods were substantially less efficient when compared with the use of viruses, particularly when used in vivo. Herein a three-step, highly efficient method of nonviral gene delivery is presented. Using this method, genes have been delivered successfully into tissues of orthopaedic importance with high-efficiency by nonviral means. Transforming growth factor-beta 1, parathyroid hormone related protein, and a marker gene were transfected into primary perichondrium and cartilage cells with efficiencies in excess of 70%. They overexpressed their cognate gene products showing efficacy of expression in a rabbit model of osteochondral defect repair. Using the same method, a marker gene was delivered into a canine model for intrasynovial flexor tendon injury and repair. This was achieved by direct gene delivery during surgery. An estimated 5 additional minutes were required during surgery to complete the transfection steps. High efficiency gene delivery was achieved in the flexor tendons, tendon sheaths, tendon pulleys, surrounding tissues, and skin. The efficiency of transfection approached 100% in the exposed superficial tissue layers and transfected cells were found several layers below the exposed tissue surfaces. The data show the potential of direct nonviral gene therapy in orthopaedics for ex vivo and in vivo applications.