George A. Ksander

The effect of platelet releasate on wound healing in animal models

The alpha granules of platelets contain growth factors that are important in wound healing. We found that a major effect of thrombin-induced human platelet releasates in animal models of wound healing is to enhance the development of granulation tissue and new connective tissue matrix. These studies provide further evidence that platelet-derived protein factors may be useful in treating full-thickness dermal wounds by increasing the rate of granulation tissue formation.

The influence of heparin on the wound healing response to collagen implants in vivo

The biologic response to fibrillar collagen (collagen) and fibrillar collagen plus heparin (collagen/heparin) implants have been compared in the rat subcutaneous and guinea pig dermal wound models. The reconstituted bovine dermal collagen implants were injected subcutaneously in rats at concentrations ranging from 18 to 30 mg/ml and in volumes ranging from 0.5 to 1.0 ml. The biologic response to the collagen implants alone was characterized by a transient invasion of a modest number of inflammatory cells within the first three days of implantation that was followed by limited fibroblast invasion into the peripheral 1/3 of the implant during the course of the next three to four weeks. Occasionally, blood vessels were observed to invade the peripheral regions of the implant. The degree (number) and extent (depth) of cell invasion were inversely related to initial collagen implant concentration. Addition of heparin (0.3-20 micrograms/mg collagen) to these implants resulted in a significant dose-dependent increase in the degree and extent of fibroblast invasion. Radiolabeling studies showed that the collagen and collagen/heparin implants were cleared from the subcutis at identical rates. Implantation of these formulations in a guinea pig dermal wound model was also performed, using a semi-occlusive wound dressing (Opsite) to maintain the implant in the wound site. The fibrillar collagen implant alone was pushed upward by developing granulation tissue at the base of the wound and served as a support for epidermal cell migration, proliferation, and differentiation as wound closure proceeded. The implant was slowly invaded and turned over as granulation tissue developed from the base and margins of the wound bed. The inclusion of heparin in these implants resulted in a significantly different pattern of wound healing. The collagen/heparin implants histologically presented a more broken-up or porous appearance following implantation, which was associated with a greater degree of penetration of developing granulation tissue into the implant itself as compared to the collagen implants. Radiolabeling studies revealed that clearance rates of implants with and without heparin from wound sites were similar, as noted in the rat subcutis. Laser doppler flowmetry studies suggested that the heparin--containing implants were more vascular than control wound sites or sites treated with collagen alone.

Skin distribution and differential expression of transforming growth factor β1 and β2

Abstract Transforming growth factor β (TGF-β) 1 and 2 have both become increasingly important in cutaneous biology, but their expression and distribution in human skin are not entirely clear. In this report, normal forearm skin from four volunteers was investigated for TGF-β1 and β2 immunostaining with antibodies that detect preferentially either cell- or matrix-associated forms of these peptides. Marked cell-associated TGF-β1 was found in the dermis, particularly around blood vessels and ducts; cellular TGF-β2 immunostaining was less prominent, and was predominantly around blood vessels. Neither TGF-β1 nor -β2 could be detected in the epidermis or epithelial structures, and the dermal matrix contained minimally detectable amounts of the two isoforms. In all cases, dermal matrix and cells contained greater amounts of TGF-β1 than TGF-β2. Previous studies have shown that both TGF-β1 and -β2 can induce dramatic increases in extracellular matrix, and both peptides have been implicated in the pathogenesis of fibrosis. We therefore investigated TGF-β1 and -β2 immunostaining in involved forearm skin of four patients with systemic sclerosis. Compared to normal skin, fibrotic specimens showed increased amounts of matrix and epidermal TGF-β1, but not TGF-β2. We conclude that TGF-β1 and -β2 expression in human skin is differentially regulated, and that their distribution is varied and complex.

Differences in the biological activities of transforming growth factor-beta and platelet-derived growth factor in vivo.

Transforming growth factor-beta 1 (TGF-beta 1 and recombinant platelet-derived growth factor-BB (rPDGF-BB) promoted an extensive, dose-dependent development of fibrous connective tissue when continuously delivered for 8 days by mini-osmotic pumps implanted subcutaneously in adult guinea pigs. Biochemical analyses demonstrated that TGF-beta 1 and rPDGF-BB stimulated dose-dependent increases in the dry weight, and protein, DNA, collagen, and glycosaminoglycan (GAG) contents of the fibrous connective tissue capsule that enveloped the pumps. The GAG/DNA mass ratio was markedly elevated by TGF-beta 1, but the collagen/DNA, protein/DNA, and collagen/protein ratios were not significantly increased. In contrast, rPDGF-BB generally decreased these mass ratios. Histological analyses suggested that this was due to the fact that rPDGF-BB induced a very cellular response with a marked influx of neutrophils and fibroblasts. TGF-beta 1 induced significantly less cellular response, which consisted primarily fibroblasts and macrophages. These results indicated that rPDGF-BB and TGF-beta 1 induced connective tissue deposition in vivo in a dose-dependent fashion, although the cellular nature of the responses as well as the structural composition of the extracellular matrices were clearly distinguishable between the two growth factors.

Transforming Growth Factors-βl and β2 Induce Synthesis and Accumulation of Hyaluronate and Chondroitin Sulfate In Vivo

Subcutaneous implantation in rats of partially purified transforming growth factor-beta (TGF-beta) derived from bovine bone induced extensive development of connective tissue with associated edema. Subcutaneous injection of pure TGF-beta 1 or TGF-beta 2 also induced connective tissue deposition in mice and guinea pigs. Sustained release of TGF-beta 1 from mini-osmotic pumps implanted subcutaneously in mature guinea pigs promoted connective tissue deposition that encapsulated the pumps. Biochemical analyses of the connective tissue capsule demonstrated that TGF-beta 1 induced a dose-dependent accumulation of glycosaminoglycans (GAGs). The GAG/DNA ratio also increased as a function of the rate of TGF-beta 1 released, suggesting that the factor increased production of GAGs per cell. Cellulose acetate gel electrophoresis of the GAGs and hydrolysis with specific glycosidases revealed that the majority of GAGs consisted of hyaluronate and chondroitin sulfate. These results demonstrate that TGF-beta 1 and TGF-beta 2 stimulate the production of not only collagenous extracellular matrix components, but also dramatically increase the in vivo synthesis of hyaluronate and chondroitin sulfate.

Exogenous transforming growth factor-β2 enhances connective tissue formation in transforming growth factor-β1—deficient, healing-impaired dermal wounds in mice

Connective tissue formation is markedly reduced in full‐thickness mouse dermal wounds that are covered with synthetic, adherent, moisture vapor—permeable membrane when compared with formation in similar but nonoccluded wounds. The transforming growth factors‐β (TGF‐β) are a family of multifunctional peptides thought to have a critical role in the regulation of development and tissue repair. Treatment with exogenous TGF‐β1 stimulated connective tissue formation in wounds covered with synthetic, adherent, moisture vapor—permeable membrane but had no effect on air‐exposed wounds, suggesting that the quantity of endogenous TGF‐β1 in wounds covered with synthetic, adherent, moisture vapor—permeable membrane was less than that in air‐exposed wounds. Immunolocalization studies with an anti‐TGF‐β1 antibody confirmed that wounds covered with synthetic, adherent, moisture vapor—permeable membrane demonstrated markedly reduced levels of endogenous extracellular, matrix‐associated TGF‐β1 as early as 24 hours after wounding. Immunoreactive TGF‐β2 was not detected. These findings suggest that endogenous TGF‐β1, but not TGF‐β2, is required for normal connective tissue formation in this model and that impaired healing is associated with low levels of TGF‐β1. Histologic analysis confirmed previous demonstrations that exogenous TGF‐β2 stimulates enhanced cellularity and connective tissue formation. Immunolocalization showed that exogenous TGF‐β2 stimulates increased expression of endogenous TGF‐β1. Northern blot analysis revealed that TGF‐β2 increased the expression of genes encoding the α1‐chain of types I and III collagens and tissue inhibitor of metalloproteinase‐1. These observations show that TGF‐β2 acts through a variety of mechanisms to stimulate repair in healing‐impaired wounds that are also deficient in endogenous TGF‐β1, but they do not distinguish between direct effects and indirect effects mediated by induced TGF‐β1.

Differential response to transforming growth factors‐β1 and β2: connective tissue deposition in animal models

The ability of transforming growth factor (TGF)‐β1 and TGF‐β2 to promote connective tissue deposition were compared in different animal models. A single subcutaneous injection of TGF‐β2 in collagen/heparin gel carrier promoted markedly more extensive development of connective tissue than TGF‐β1 at the site of injection in both neonatal and adult mice. Both TGF‐β1 and TGF‐β2 promoted deposition of dense and well‐vascularized connective tissue matrix infiltrated with macrophages and fibroblasts. However, the results of immunohistochemical analyses suggested that TGF‐β2 promoted an accumulation of more macrophages in the connective tissue than TGF‐β1. Similar differences in the extent of connective tissue development were observed in neonatal mice when these factors were administered as a solution, without the collagen/heparin gel carrier. TGF‐β2 was also more potent than TGF‐β1 in domestic pigs. However, in guinea pigs, TGF‐β1 promoted more extensive connective tissue development than TGF‐β2. These results suggest that the differential connective tissue response to TGF‐β1 and TGF‐β2 is species dependent. However, the differences in the physical and chemical properties of these factors may account in part for the differential response as well.