Microencapsulated cells genetically modified to overexpress human transforming growth factor-beta1: viability and functionality in allogeneic and xenogeneic implant models.

This study explores the suitability of using encapsulated genetically modified fibroblasts for orthopedic tissue engineering by examining cell survival and persistence of human transforming growth factor-beta (hTGF-beta) overexpression in xenogeneic and allogeneic implant models. Human wild-type fibroblasts, modified to produce a latent form of hTGF-beta, and murine mutant-type fibroblasts, engineered to release a constitutively active form of hTGF-beta, were encapsulated separately in Ca2+ -alginate microcapsules. Following a percentage viability assessment by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test, microcapsules were implanted into either the subcutaneous or intraperitoneal cavities of mice. Explanted encapsulated cells were characterized for percentage viability and subjected to a release study and a viability test 1 week and 3 weeks following implantation, a time frame consistent with the requirement for orthopedic tissue engineering application of this growth factor. On average, percentage viabilities of encapsulated cells were 64%at implantation, 52% at explantation, and 56%after 1 week following either 1- or 3-week explantation. hTGF-beta release declined following in vivo implantation, more so for xenogeneic than allogeneic models, but remained in the clinically attractive range of 2 to 30 ng/(10(6) implanted cells 24 h). This technical platform for hTGF-beta is very encouraging for cartilage regeneration using orthopedic tissue engineering, and further evaluation is warranted.

Upregulation of TGF-beta1 expression may be necessary but is not sufficient for excessive scarring.

Transforming growth factor beta 1 (TGF-beta1) upregulation has been implicated in hypertrophic scars and keloids, but it is unclear if it is the cause or an effect of excessive scar formation. In this study, we overexpressed TGF-beta1 in fibroblasts and characterized its role. Normal human dermal fibroblasts were genetically modified to overexpress TGF-beta1 as the wild-type latent molecule or as a mutant constitutively active molecule. TGF-beta1 secretion was measured, as were the effects of TGF-beta1 upregulation on cell proliferation, expression of smooth muscle cell alpha actin (SMC alpha-actin) and ability to contract collagen lattices. Fibroblasts were implanted intradermally into athymic mice and tissue formation was analyzed over time by histology and immunostaining. Gene-modified fibroblasts secreted approximately 20 times the TGF-beta1 released by control cells, but only cells expressing mutant TGF-beta1 secreted it in the active form. Fibroblasts expressing the active TGF-beta1 gene had increased levels of SMC alpha-actin and enhanced ability to contract a collagen lattice. After intradermal injection into athymic mice, only fibroblasts expressing active TGF-beta1 formed "keloid-like" nodules containing collagen, which persisted longer than implants of the other cell types. We conclude that upregulation of TGF-beta1 by fibroblasts may be necessary, but is not sufficient for excessive scarring. Needed are other signals to activate TGF-beta1 and prolong cell persistence.

In vitro characterization of TGF-beta1 release from genetically modified fibroblasts in Ca(2+)-alginate microcapsules.

This study was undertaken to develop an in situ source of transforming growth factor-beta1 (TGF-beta1), one of several molecules potentially useful for a tissue-engineered bioartificial cartilage. Primary human fibroblasts and murine NIH 3T3 cells were genetically modified via viral transfection to express human TGF-beta1. Two viral constructs were used, one expressing a gene encoding for the latent and the other for the constitutively active form of the growth factor. Unmodified cells served as controls. Four genetically modified cohorts and two controls were separately encapsulated in a 1.8% alginate solution using a vibrating nozzle and 0.15M calcium chloride crosslinking bath. Diameter of the spherical capsules was 410 +/- 87 microm. In vitro release rate measured over 168 hours varied with cell types and ranged from 2-17 pg/(milligram of capsules x 24 h) or 2-17 ng/(10(6) cells x 24 h). None of the formulations exhibited a large initial bolus release. Even when serum-supplemented medium was not replenished, cell viabilities remained over 55% after 1 week for all cell types. Microencapsulated genetically modified cells were capable of a constitutive synthesis and delivery of biologically significant quantity of TGF-beta1 for at least 168 hours and thus are of potential utility for artificial cartilage and other orthopedic tissue engineering applications.