Mathew Varkey

Growth factor delivery for bone tissue repair: an update

Growth factors (GFs) are endogenous proteins capable of acting on cell-surface receptors and directing cellular activities involved in the regeneration of new bone tissue. The specific actions and long-term effects of GFs on bone-forming cells have resulted in exploration of their potential for clinical bone repair. The concerted efforts have led to the recent approval of two GFs, bone morphogenetic protein-2 and osteogenic protein-1, for clinical bone repair, and human parathryroid hormone (1-34) for augmentation of systemic bone mass. This review provides a selective summary of recent (2001 – 2004) attempts for GF delivery in bone tissue regeneration. First, a summary of non-human primate studies involving local regeneration and repair is provided, with special emphasis on the range of biomaterials used for GF delivery. Next, efforts to administer GFs for systemic augmentation of bone tissue are summarised. Finally, an alternative means of GF delivery, namely the delivery of genes coding for osteogenic proteins, rather than the delivery of the proteins, is summarised from rodent models. To conclude, future avenues of research considered promising to enhance the clinical application of GFs are discussed.

In vitro osteogenic response of rat bone marrow cells to bFGF and BMP-2 treatments.

Basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) are actively pursued for stimulation of bone formation. To assess their promise for systemic therapy of osteoporosis, we ascertained the effects of bFGF and BMP-2 on bone marrow cells in vitro. Bone marrow cells were obtained from young (8 weeks) and adult (32 weeks) rats by femoral aspiration and were exposed to osteogenic medium (ie, basal medium with 10 mM β-glycerolphosphate and 100 nM dexamethasone) containing the growth factors. The cell viability in osteogenic medium was reduced after 3 weeks but not if the concentration of β-glycerolphosphate/dexamethasone was reduced to 3 mM/30 nM. Unlike BMP-2, bFGF at 2-50 ng/mL was capable of enhancing long-term cell viability. Continuous treatment of bone marrow cells for 3 weeks resulted in dose-dependent stimulation of mineralization by BMP-2, but not by bFGF, whose activity was optimal at 2-10 ng/mL. To explore the effect of short-term exposure, bone marrow cells were treated with growth factors for 1 week and subsequent mineralization was investigated. BMP-2 exposure increased the extent of mineralization, but bFGF was not effective after the short exposure. We concluded bFGF was more potent (ie, required lower concentration) for stimulating osteogenic parameters, but BMP-2 effects were lasting on the bone marrow cells.

Small leucine-rich proteoglycans, decorin and fibromodulin, are reduced in postburn hypertrophic scar.

Small leucine‐rich proteoglycans (SLRPs) are extracellular matrix molecules that regulate collagen fibrillogenesis and inhibit transforming growth factor‐β activity; thus, they may play a critical role in wound healing and scar formation. Hypertrophic scarring is a dermal form of fibroproliferative disorders, which occurs in over 70% of burn patients and leads to disfigurement and limitations in function. By understanding the cellular and molecular mechanisms that lead to scarring after injury, new clinical therapeutic approaches can by developed to minimize abnormal scar formation in hypertrophic scarring and other fibroproliferative disorders. To study the expression and localization of SLRPs with connective tissue cells in tissue immunohistochemistry, immunofluorescence staining, immunoblotting, and reverse‐transcription polymerase chain reaction were used in normal skin and hypertrophic scar (HTS). In normal skin, there was more decorin and fibromodulin accumulation in the superficial layers than in the deeper dermal layers. The levels of decorin and fibromodulin were significantly lower in HTS, whereas biglycan was increased when compared with normal skin. There was an increased expression of biglycan, fibromodulin, and lumican in the basement membrane and around basal epithelial cells. In contrast, these proteoglycans were absent or weakly expressed in HTS. The findings suggest that down‐regulation of SLRPs after wound healing in deep injuries to the skin plays an important role in the development of fibrosis and HTS.

Reduced Decorin, Fibromodulin, and Transforming Growth Factor-β3 in Deep Dermis Leads to Hypertrophic Scarring

Hypertrophic scar (HTS) occurs after injuries involving the deep dermis, while superficial wounds (SWs) to the skin heal with minimal or no scarring. The levels of transforming growth factor (TGF)-&bgr;1 and small leucine-rich proteoglycans (SLRPs) with fibroblast subtype and function may influence the development of HTS. The aim of this study was to characterize the expression and localization of factors that regulate wound healing including SLRPs, TGF-&bgr;1, and TGF-&bgr;3 in an experimental human SW and deep wound (DW) scar model including fibroblasts from superficial and deep layers of normal dermis. A 6-cm horizontal dermal scratch experimental wound was created, which consisted of progressively deeper wounds that were superficial at one end (0–0.75 mm deep) and deep (0.75–3 mm deep) at the other end, located on the anterior thigh of an adult male. Immunofluorescence staining, immunoblotting, reverse transcription polymerase chain reaction, and flow cytometry were performed to analyze the cellular and molecular differences between the SW scar and DW scar as well as fibroblasts isolated from superficial layer (L1) and deep layer (L5) of normal dermis. Comparing SWs and L1 fibroblasts, the expression of decorin, fibromodulin, and TGF-&bgr;3 was considerably lower than in DWs and L5 fibroblasts; however, TGF-&bgr;1 was higher in the deeper dermal wounds. When compared with L1 fibroblasts, L5 fibroblasts had lower Thy-1 immunoreactivity and significantly higher expression of TGF-&bgr; receptor type II. Decreased antifibrotic molecules in matrix of deep dermis of the skin and the unique features of the associated fibroblasts including an increased sensitivity to TGF-&bgr;1 stimulation contribute to the development of HTS after injuries involving the deep dermis.

Differential collagen–glycosaminoglycan matrix remodeling by superficial and deep dermal fibroblasts: Potential therapeutic targets for hypertrophic scar

Skin substitutes are the preferred treatment option in the case of extensive skin loss following burns or other injuries. Among skin substitutes, cultured skin substitutes containing autologous fibroblasts and keratinocytes on collagen-glycosaminoglycan (C-GAG) matrix are most preferred for wound repair. A significant negative outcome of wound healing is hypertrophic scarring (HTS), a dermal fibroproliferative disorder, that leads to considerable morbidity. To examine the role of superficial and deep dermal fibroblasts in HTS, and determine if they differentially remodel C-GAG matrices, fibroblasts were isolated from superficial and deep dermis of lower abdominal tissue of abdominoplasty patients and cultured on C-GAG matrices for four weeks. Over time, deep fibroblasts contracted and stiffened the matrices significantly more and decreased their ultimate tensile strength compared to superficial fibroblasts. Differential remodeling of C-GAG matrices by fibroblasts obtained from different locations of the same organ has not been reported before. Deep fibroblasts were found to express significantly more osteopontin, angiotensin-II, peroxisome proliferator-activated receptor (PPAR)-α, and significantly less tumor necrosis factor-α, PPAR-β/δ, PPAR-γ, and the proteoglycan, fibromodulin compared to superficial fibroblasts. These molecular targets could potentially be used in therapeutic strategies for treatment of HTS.

Deep dermal fibroblasts refractory to migration and decorin-induced apoptosis contribute to hypertrophic scarring.

Hypertrophic scar (HTS) represents the dermal equivalent of fibroproliferative disorders. Fibroblasts from the deep dermis are implicated in the development of HTS after injuries that involve deeper areas of the skin. However, fibroblasts that reside in the superficial layer of the skin show antifibrotic properties, and injuries limited to this area heal with little or no scarring. Previously, cellular and molecular characteristics of superficial fibroblasts and deep dermal fibroblasts that may influence HTS formation were analyzed. In this study, differences in cellular behavior between superficial fibroblasts and deep dermal fibroblasts that may also affect the development of HTS or tissue fibrosis were further characterized. Immunostaining and migration, adhesion, apoptosis, and cell viability assays were performed in fibroblasts from the superficial and deep dermis. Reverse-transcription polymerase chain reaction was used to examine the gene expression of molecules involved in cell death after treatment of fibroblasts with decorin. When compared with superficial fibroblasts, deep dermal fibroblasts showed lower migration rates. Although all the fibroblasts tested showed no difference in adhesion to fibronectin, superficial fibroblasts demonstrated increased apoptotic and dead cells when treated with decorin. Decorin resulted in a significant increase in the expression of apoptosis markers, histone-1, caspase-1, caspase-8, and p53 in superficial fibroblasts when compared with deep dermal fibroblasts. Taken together, the findings suggest that reduced migration, lack of decorin, and resistance of deep dermal fibroblasts to decorin-induced apoptosis may result in hypercellularity in injuries involving the deep dermis, leading to deposition of excess extracellular matrix and HTS formation.

Advances in Skin Substitutes—Potential of Tissue Engineered Skin for Facilitating Anti-Fibrotic Healing

Skin protects the body from exogenous substances and functions as a barrier to fluid loss and trauma. The skin comprises of epidermal, dermal and hypodermal layers, which mainly contain keratinocytes, fibroblasts and adipocytes, respectively, typically embedded on extracellular matrix made up of glycosaminoglycans and fibrous proteins. When the integrity of skin is compromised due to injury as in burns the coverage of skin has to be restored to facilitate repair and regeneration. Skin substitutes are preferred for wound coverage when the loss of skin is extensive especially in the case of second or third degree burns. Different kinds of skin substitutes with different features are commercially available; they can be classified into acellular skin substitutes, those with cultured epidermal cells and no dermal components, those with only dermal components, and tissue engineered substitutes that contain both epidermal and dermal components. Typically, adult wounds heal by fibrosis. Most organs are affected by fibrosis, with chronic fibrotic diseases estimated to be a leading cause of morbidity and mortality. In the skin, fibroproliferative disorders such as hypertrophic scars and keloid formation cause cosmetic and functional problems. Dermal fibroblasts are understood to be heterogeneous; this may have implications on post-burn wound healing since studies have shown that superficial and deep dermal fibroblasts are anti-fibrotic and pro-fibrotic, respectively. Selective use of superficial dermal fibroblasts rather than the conventional heterogeneous dermal fibroblasts may prove beneficial for post-burn wound healing.

In Vitro Osteogenic Induction Of Human Gingival Fibroblasts For Bone Regeneration

Background And Objective: Periodontitis is an inflammatory disease causing bone loss, and is a primary cause of tooth loss. Gingival fibroblasts are readily available with minimal donor site morbidity and may be ideal for tissue engineering efforts in regenerating lost alveolar bone. Dexamethasone (Dex) is commonly employed for in vitro osteogenic induction of a variety of cells, but its effect on human gingival fibroblasts (HGF) is still controversial. Therefore, the aim of our study was to investigate the osteogenic differentiation of HGF following Dex treatment. Methods: Cultured HGFs were exposed to osteogenic medium containing a wide range of Dex concentrations (0.01-10 µM). The osteogenic phenotype was assessed based on changes in alkaline phosphatase (ALP) activity, the mRNA expression of selected extracellular matrix proteins critical for mineralization and the extent of extracellular mineralization (Von Kossa staining and Ca-content). Results: All assays showed a consistent and maximal osteogenic effect of Dex on HGF at 0.1 and 0.5 µM (weeks 3 and 4), as evidenced by significant osteopontin and osteocalcin expression and mineralization. Longer cultures (week 4) also yielded positive osteogenic effect of Dex at 0.01 µM. Moreover, ALP activity was significantly stimulated at 0.1 and 0.5 µM Dex initially after one week, but ALP was subsequently reduced under Dex. Higher Dex concentrations caused down regulation of osteogenic effects observed at the optimal (0.1-0.5 µM) concentrations. Conclusion: Under appropriate osteogenic conditioning, Dex treated HGFs could be a potential source of cells for cell-based therapy for periodontal bone regeneration.

Fibrotic remodeling of tissue-engineered skin with deep dermal fibroblasts is reduced by keratinocytes.

Two-thirds of burn patients with deep dermal injuries are affected by hypertrophic scars, and currently, there are no clinically effective therapies. Tissue-engineered skin is a very promising model for the elucidation of the role of matrix microenvironment and biomechanical characteristics and could help in the identification of new therapeutic targets for hypertrophic scars. Conventionally, tissue-engineered skin is made of heterogeneous dermal fibroblasts and keratinocytes; however, recent work has shown that superficial and deep dermal fibroblasts are antifibrotic and profibrotic, respectively. Furthermore, keratinocytes are believed to regulate the development and remodeling of fibrosis in skin. This study aimed to assess the influence of keratinocytes and layered fibroblasts on the characteristics of tissue-engineered skin. Layered fibroblasts and keratinocytes isolated from superficial and deep dermis and epidermis, respectively, of the lower abdominal tissue were independently co-cultured on collagen-glycosaminoglycan scaffolds, and the resulting tissue-engineered skin was assessed for differences in tissue remodeling based on the underlying specific dermal fibroblast subpopulation. Collagen production by deep fibroblasts but not by superficial fibroblasts was significantly reduced upon co-culture with keratinocytes. Also, keratinocytes in the tissue-engineered skin resulted in significantly reduced expression of profibrotic connective tissue growth factor and fibronectin, and increased expression of the antifibrotic matrix metalloproteinase-1 by deep fibroblasts but not by superficial fibroblasts. Tissue-engineered skin made of deep fibroblasts and keratinocytes had lower levels of small proteoglycans, decorin, and fibromodulin, and higher levels of large proteoglycan, versican, compared to tissue-engineered skin made of superficial fibroblasts and keratinocytes. Tissue-engineered skin made of deep fibroblasts and keratinocytes had lower expression of transforming growth factor (TGF)-α, interleukin (IL)-1, and keratinocyte growth factor but higher expression of platelet-derived growth factor and IL-6, compared to tissue-engineered skin made of superficial fibroblasts and keratinocytes. Furthermore, co-culture with keratinocytes reduced TGF-β1 production of deep but not superficial fibroblasts. Additionally, keratinocytes reduced the differentiation of deep fibroblasts to myofibroblasts in tissue-engineered skin constructs, but not that of superficial fibroblasts. Taken together, keratinocytes reduce fibrotic remodeling of the scaffolds by deep dermal fibroblasts. Our results therefore demonstrate that tissue-engineered skin made specifically with a homogeneous population of superficial fibroblasts and keratinocytes is less fibrotic than that with a heterogeneous population of fibroblasts and keratinocytes.

The effect of keratinocytes on the biomechanical characteristics and pore microstructure of tissue engineered skin using deep dermal fibroblasts

Fibrosis affects most organs, it results in replacement of normal parenchymal tissue with collagen-rich extracellular matrix, which compromises tissue architecture and ultimately causes loss of function of the affected organ. Biochemical pathways that contribute to fibrosis have been extensively studied, but the role of biomechanical signaling in fibrosis is not clearly understood. In this study, we assessed the effect keratinocytes have on the biomechanical characteristics and pore microstructure of tissue engineered skin made with superficial or deep dermal fibroblasts in order to determine any biomaterial-mediated anti-fibrotic influences on tissue engineered skin. Tissue engineered skin with deep dermal fibroblasts and keratinocytes were found to be less stiff and contracted and had reduced number of myofibroblasts and lower expression of matrix crosslinking factors compared to matrices with deep fibroblasts alone. However, there were no such differences between tissue engineered skin with superficial fibroblasts and keratinocytes and matrices with superficial fibroblasts alone. Also, tissue engineered skin with deep fibroblasts and keratinocytes had smaller pores compared to those with superficial fibroblasts and keratinocytes; pore size of tissue engineered skin with deep fibroblasts and keratinocytes were not different from those matrices with deep fibroblasts alone. A better understanding of biomechanical characteristics and pore microstructure of tissue engineered skin may prove beneficial in promoting normal wound healing over pathologic healing.