Deep dermal fibroblast profibrotic characteristics are enhanced by bone marrow–derived mesenchymal stem cells

Hypertrophic scars are a significant fibroproliferative disorder complicating deep injuries to the skin. We hypothesize that activated deep dermal fibroblasts are subject to regulation by bone marrow–derived mesenchymal stem cells (BM‐MSCs), which leads to the development of excessive fibrosis following deep dermal injury. We found that the expression of fibrotic factors was higher in deep burn wounds compared with superficial burn wounds collected from burn patients with varying depth of skin injury. We characterized deep and superficial dermal fibroblasts, which were cultured from the deep and superficial dermal layers of normal uninjured skin obtained from abdominoplasty patients, and examined the paracrine effects of BM‐MSCs on the fibrotic activities of the cells. In vitro, deep dermal fibroblasts were found higher in the messenger RNA (mRNA) levels of type 1 collagen, alpha smooth muscle actin, transforming growth factor beta, stromal cell–derived factor 1, and tissue inhibitor of metalloproteinase 1, an inhibitor of collagenase (matrix metalloproteinase 1). As well, deep dermal fibroblasts had low matrix metalloproteinase 1 mRNA, produced more collagen, and contracted collagen lattices significantly greater than superficial fibroblasts. By co‐culturing layered fibroblasts with BM‐MSCs in a transwell insert system, BM‐MSCs enhanced the fibrotic behavior of deep dermal fibroblasts, which suggests a possible involvement of BM‐MSCs in the pathogenesis of hypertrophic scarring.     

Persistent Ischemia Impairs Myofibroblast Development in Wound Granulation Tissue a New Model of Delayed Wound Healing

Skin substitutes are slowly finding a position in the treatment of burns, scar reconstructions and chronic wounds. Some of the substitutes consist of extracellular matrix replacement material only, such as Integra and Alloderm; some include allogeneic cells (Dermagraft, Appligraf). The ideal skin substitute has not been developed yet, since none of the presently available products can ultimately prevent scar formation.
Study of the role of autologous fibroblasts in the healing process might give further insight into the scarring process and eventually lead to improved skin substitutes.
We compared wound reepithelialization of experimental wounds treated with proliferating keratinocytes and a dermal substitute with either dermal fibroblasts, adipose tissue derived fibroblasts or no fibroblasts. We also investigated the rate of keratinocyte migration of human skin equivalents cultured in vitro in the presence of dermal or adipose tissue derived fibroblasts.
We reached successful wound closure in 8 days with transfer of proliferating keratinocytes on a dermal substitute seeded with dermal fibroblasts. However, the wounds treated with substitutes which contained adipose tissue derived fibroblasts or no fibroblasts at all were not closed even after 21 days.
Keratinocytes seeded onto collagen lattices populated with either dermal or fat‐derived fibroblasts showed similar findings: a retarded migration and/or proliferation of keratinocytes on the collagen lattices with fat‐derived fibroblasts. The collagen lattices populated with fat‐derived fibroblasts also showed a marked contraction, up till 50% of the original area.
In both models, more alpha‐smooth muscle actin positive cells were found in the fibroblast population from adipose origin.
We conclude that epidermal regeneration is negatively influenced by the presence of fat‐derived fibroblasts in a dermal matrix; possibly, myofibroblasts play a role in this.     

Breast capsule contracture: Is fibroblast activity associated with severity?

Factors responsible for breast capsule contracture remain elusive. Using an in vitro model of wound contraction, the fibroblast-populated collagen lattice (FPCL), breast capsule fibroblasts and control dermal fibroblasts from ten patients were analyzed. Comparison was made to determine (1) if the activity of dermal fibroblasts on a collagen lattice correlated with the activity of breast capsule fibroblasts or if capsular fibroblasts are unique, and (2) if the degree of fibroblast-driven collagen contraction correlated with clinical severity of breast capsule contracture. If so, a preoperative predictor of breast capsule contracture would be available. Dermal fibroblasts and breast capsule fibroblasts were cultured and mixed with media and collagen to form a matrix, and then the degree of lattice contraction was measured. A correlation between breast capsule fibroblasts and control dermal fibroblasts with respect to collagen matrix contraction was confirmed. Collagen lattice contraction coordinated by fibroblasts derived from breast capsules did not correlate with clinical severity of capsular contracture. These results indicate that the degree of breast capsule contracture can not be predicted by fibroblast activity alone. An interaction between inflammatory cells, extracellular matrix, and fibroblasts is hypothesized. Further work is needed to delineate the mechanisms responsible for breast capsule contracture.     

Dermal regeneration in native non-cross-linked collagen sponges with different extracellular matrix molecules

Collagenous dermal templates can prevent scarring and wound contraction in the healing of full-thickness defects. In a porcine wound model, full-thickness wounds were substituted by reconstituted and native collagen sponges in combination with autologous split-skin mesh grafts and covered with a semipermeable wound membrane. Native collagen sponges were also linked with either hyaluronic acid, elastin, or fibronectin. Reconstituted collagen matrixes, composed of cross-linked small collagen fibrils, disintegrated within a week and did not contribute to dermal regeneration, whereas native collagen matrixes, composed of intact collagen fibers, disintegrated within 2 weeks and did contribute to dermal regeneration. Addition of extracellular matrix proteins retarded the disintegration to 4 weeks. However, fibronectin-treated matrixes caused aberrant epithelization. When hyaluronic acid was added, matrixes were invaded by more fibroblasts and myofibroblasts. This process correlated with fibrosis and wound contraction. In contrast, the native collagen/elastin matrix reduced the amount of fibroblasts and myofibroblasts. This latter matrix resulted in optimal dermal regeneration and little wound contraction.     

High yields of autologous living dermal equivalents using porcine gelatin microbeads as microcarriers for autologous fibroblasts

Permanent skin replacement requires a dermal component to ensure adequate long-term graft stability and to prevent wound contraction. This study was to construct a bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous living dermal equivalents on a large scale. Autologous fibroblasts were isolated from split-thickness skin biopsy from a leg ulcer patient, inoculated onto macroporous porcine gelatin microbeads, and incubated in a bioreactor (Cellspin) in serum-free fibroblast growth medium or in DMEM medium containing 10% fetal calf serum (FCS). Fibroblasts rapidly adhered to and actively proliferated on the microbeads in the bioreactor in both serum-free and serum-containing medium. MTT assay showed the number of fibroblasts on the microbeads reached up to 5.3- or 4.0-fold the cells seeded in DMEM medium containing 10% FCS or serum-free medium, respectively. When removed from Bio-MCCS and cultured under static conditions, fibroblasts were able to leave the microbeads and proliferate to confluence on the bottom of tissue culture flasks. When stored at room temperature in DMEM containing 10% FBS, fibroblast cultured on the microbeads retained highest viabilities for at least 3 weeks, up to 82% of originals. This Bio-MCCS using porcine gelatin microbeads as carriers for fibroblasts offers a new option of mass production of autologous living dermal equivalents.     

In vitro formation and expansion of cysts derived from human renal cortex epithelial cells.

Acquired renal cysts derive from terminally differentiated tubular epithelium in adults as a consequence of increased epithelial cell proliferation, fluid accumulation and extracellular matrix remodelling. To understand better how human epithelial cysts may be initiated and progressively expand, cells from primary cultures of normal human adult renal cortex were dispersed in polymerized type I collagen. The transparent matrix permitted repeated observation by light microscopy of cyst formation from individual renal cells. The cyst cells reacted strongly with distal nephron histochemical markers (cytokeratin antibodies AE1/AE3, epithelial membrane antigen, and Arachis hypogaea lectin) but inconsistently or not at all to markers of proximal tubules (Tetragonolobus purpureas lectin and Phaseolus vulgaris erthroagglutinin lectin). The number of spherical, fluid-filled epithelial cysts that developed in a standardized microscope field quantified cyst initiation. Cyst progression was determined from the increase in the diameter (surface area) of cysts and represents a hyperplastic event. EGF or TGF alpha, were required in serum-free defined medium to cause cysts to develop from individual epithelial cells dispersed in the matrix; insulin was required as a co-factor. The EC50 for EGF was approximately 0.1 ng/ml, and for insulin 1 microgram/ml. Early cultures of normal cortex formed cysts more efficiently when dispersed in collagen matrix than cells passaged several times before suspension in the gel. Agonists of adenylate cyclase (PGE1, AVP, VIP, PTH, forskolin, cholera toxin), methylisobutylxanthine, and 8-Br-cAMP, though incapable of causing cyst formation alone in defined medium, enhanced cyst initiation and progression in the presence of EGF and insulin. Angiotensin II, TNF alpha, beta-estradiol, and pertussis toxin had no effect in the absence or presence of EGF and insulin. Pertussis toxin inhibited cyst initiation and expansion caused by EGF and forskolin but potentiated cyst initiation and expansion caused by EGF and PGE1. Cyst formation and expansion were inhibited by TGF beta 1 and 2-chloroadenosine. Polarized monolayers of human renal cortical cells grown on permeable membranes were used to independently quantify the effects of agonists on the net secretion of solute and water from the basolateral to the apical surface of the cells. PGE1, forskolin, and 8-Br-cAMP stimulated net fluid secretion that was sustained for several days; EGF enhanced forskolin-stimulated fluid secretion. We conclude that the formation and expansion of in vitro cysts derived from solitary human cortex cells depends on the coordinated interplay between cellular proliferation and fluid secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of a collagen matrix containing epidermal growth factor on wound contraction

Excessive wound contraction is known to lead to pathological wound contracture. Using a rabbit model, we applied a bovine type I collagen matrix sponge as a dermal substitute and human epidermal growth factor to full-thickness excisional wounds. Wound contraction was assessed 14 and 28 days after wounding. It was found that both collagen matrix and epidermal growth factor significantly inhibited wound contraction ( p < 0.001) in all wounds treated with collagen matrix alone or treated with 0.1 and 1 µg of epidermal growth factor 28 days after wounding. Interestingly, the combination of collagen matrix with epidermal growth factor strongly inhibited wound contraction over matrix alone ( p < 0.01 on day 28). Histological analyses showed a regular horizontal arrangement of collagen fibers in the dermis under wounds treated with these substances but not under untreated wounds. Furthermore, using a fibroblast-populated collagen gel, the direct inhibitory effect of epidermal growth factor on gel contraction by fibroblasts was also observed. Collagen gels without stimulation contracted to 29.5 ± 0.6% of their original size, as determined 6 days after culturing. At 3 days or more, epidermal growth factor inhibited collagen gel contraction by fibroblasts (after 6 days: 34.2 ± 1.8%, p > 0.05; 36.5 ± 2.8%, p < 0.05; and 39.8 ± 2.1%, p < 0.001 at 1, 10, and 100 ng/ml of epidermal growth factor, respectively). In conclusion, collagen matrix and epidermal growth factor, particularly in combination, may be useful in the prevention of wound contracture.     

Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels

OBJECTIVE: Neovascularization, angiogenesis, and collagen constriction are essential for wound healing. We tested whether vascular endothelial growth factor-C (VEGF-C) can promote collagen constriction, capillary sprouting (angiogenesis), and invasion/migration of bone marrow-derived endothelial progenitor cells into collagen (vasculogenesis). METHODS: We used a recently characterized three-dimensional collagen matrix assay with either monolayers of human dermal microvascular endothelial cells (HMVECs) or bone marrow-derived endothelial progenitor cells (BMD EPCs), obtained from Tie-2 LacZ transgenic mice, overlaid with an acellular layer and then a cellular layer of collagen embedded with fibroblasts, that were nontransduced or transduced with either LacZ adenoviral vector (Ad5) or VEGF-C/Ad5. The ability of VEGF-C to enhance fibroblast-mediated collagen constriction was measured, and gels overlying HMVECs or BMD EPCs were co-cultured, harvested, and assayed for HMVEC migration, sprouting, and capillary-like formation; gels containing BMD EPCs were assayed for EPC invasion/migration into the collagen extracellular matrix. RESULTS: VEGF-C significantly increased collagen constriction and formation of capillary-like structures with true lumina (P < .05) assessed by von Willebrand factor and VEGF receptor-2 immunoassaying. VEGF-C induced a significant increase in HMVEC migration, tubular polarization, and branching sprouts associated with a significant up-regulation of membrane type 1 matrix metalloproteinase (MT1-MMP) ( P < .05). Fibroblasts were necessary to support BMD-EPC invasion/migration from the monolayer into the collagen. Moreover, fibroblasts overexpressing VEGF-C significantly enhanced EPC invasion/migration ( P < .05) into the extracellular matrix by two-fold, and this effect could not be achieved with equivalent levels of exogenous VEGF-C in the absence of fibroblasts. The addition of a soluble VEGF-C competitor protein only partially inhibited these responses, reducing the EPCs by three-fold, but significant numbers of EPCs still invaded/migrated into the extracellular matrix, suggesting that other fibroblast-specific signals also contribute to the vasculogenic response. CONCLUSION: Fibroblast-specific expression of VEGF-C promotes collagen constriction by fibroblasts and enhances microvascular endothelial cell migration, branching, and capillary sprouting in association with up-regulating MT1-MMP expression. Fibroblasts are necessary for BMD EPC invasion/migration into collagen, and their overexpression of VEGF-C enhances this fibroblast-mediated vasculogenic effect. Collectively, these findings suggest a role for VEGF-C in multiple biologic steps required for wound healing (angiogenesis, vasculogenesis, and collagen constriction). CLINICAL RELEVANCE: Ischemic wound healing remains an unsolved problem with no previously identified molecular target for therapeutic intervention. This study demonstrates that VEGF-C overexpression by fibroblasts stimulates multiple biologic processes known to impact wound healing, such as collagen constriction, capillary sprouting, and EPC invasion and migration through extracellular matrix. Most ischemic wounds fail to heal and frequently lead to major limb amputation. Available cytokine ointments are ineffective, and revascularization is often not technically feasible. Even when these procedures are accomplished, many ischemic wounds frequently still do not heal because of multifactorial tissue level impairments in the fibroblastic and neovascularization responses at the wound base. Our findings identify an important role for two novel tissue level targets, dermis-derived fibroblasts and VEGF-C, in collagen constriction, angiogenesis, and postnatal vasculogenesis from BMD EPCs. Thus the findings are particularly relevant to the unsolved clinical problem of ischemic wound healing.     

组织工程化人工复合皮肤的构建

目的 为皮肤缺失的移植提供具有生物活性的表皮、真皮的组织工程化人工复合皮肤。方法 将体外传代培养的表皮角朊细胞和真皮成纤维细胞分别接种在冷冻干燥及戊二醛交联的I型胶原基质网架的两侧,在液面下培养1周后,改为气－液界面培养,动态观测人工皮肤光镜下组织学形态及电镜下超微结构。结果 人工复合皮肤具有表皮、真皮双层结构。培养过程中表皮逐步增殖、分化、发育,形成基底层、棘细胞层、颗粒层和角质层;真皮基质网架渐渐降解,并逐步被增殖的成纤维细胞及其所分泌的细胞外基质所取代,完成真皮构建。结论 利用组织工程技术体外构建人工复合皮肤,可用于皮肤缺失者的自体移植治疗,体外构建的复合人工皮肤可从根本上解决自体供皮不足,且细胞源于自体皮肤,排除了发生免疫排斥反应和疾病传播的风险,具有安全、有效、实用等优点。     

Human bone marrow-derived cells: An attractive source to populate dermal substitutes

We have previously shown the importance of dermal fibroblasts within skin substitutes for promoting the emergence of a functional neodermis after grafting in humans. However, the use of fibroblasts from sources other than the dermis needs to be evaluated for patients with extensive skin loss. Here we examined the capacity of human bone marrow-derived cells (BMDCs), selected for their ability to adhere to plastic culture dishes, to behave like human dermal fibroblasts when incorporated within a 3D in vitro reconstructed tissue that promotes dermal fibroblast differentiation. Like dermal fibroblasts, BMDCs contracted a collagen matrix and were growth regulated by the matrix environment. They had the same shape and their nuclei had the same form factor as dermal fibroblasts. In addition, both cell types expressed desmin and vimentin but not α-smooth muscle actin. BMDCs deposited collagen types I and III, and fibrillin-1 with similar efficiency to dermal fibroblasts. In addition, BMDCs have the potential to regulate this deposition, as they produced metalloproteinases (MMP1, MMP2, and MMP9) and metalloproteinase inhibitors (TIMP1) very similarly to dermal fibroblasts. BMDCs can thus be induced to express functions resembling those of dermal fibroblasts, including those involved in the wound healing process.     

Tissue engineered artificial skin composed of dermis and epidermis

Abstract: We made an artificial skin comprised of a stratified layer of keratinocytes and a dermal matrix with a type I collagen containing fibroblasts. In this work, we showed keratinocyte behavior under primary culture, gel contractions varying with concentration of collagen solution, and cell growth plots in the collagen gel. The optimum behavior of dermal equivalent could be obtained using 3.0 mg/ml collagen solution and attached gel culture. The attached gel culture had a jumping effect of growth factor on cell growth at the lag phase. To develop the artificial skin, 1× 10⁵ cells/cm² of keratinocytes were cultured on the dermal equivalent at air-liquid interface. Finally, to overcome the problem that artificial skin of collagen gel was torn easily during suturing of grafting, we prepared histocompatible collagen mesh and attached the mesh to the bottom of the gel. Cultured artificial skins were successfully grafted onto rats.     

Modeling the effects of transforming growth factor-β on extracellular matrix alignment in dermal wound repair

We present a novel mathematical model for collagen deposition and alignment during dermal wound healing, focusing on the regulatory effects of transforming growth factor-beta (TGFbeta.) Our work extends a previously developed model which considers the interactions between fibroblasts and an extracellular matrix composed of collagen and a fibrin based blood clot, by allowing fibroblasts to orient the collagen matrix, and produce and degrade the extracellular matrix, while the matrix directs the fibroblasts and control their speed. Here we extend the model by allowing a time varying concentration of TGFbeta to alter the properties of the fibroblasts. Thus we are able to simulate experiments which alter the TGFbeta profile. Within this model framework we find that most of the known effects of TGFbeta, i.e., changes in cell motility, cell proliferation and collagen production, are of minor importance to matrix alignment and cannot explain the anti-scarring properties of TGFbeta. However, we find that by changing fibroblast reorientation rates, consistent with experimental evidence, the alignment of the regenerated tissue can be significantly altered. These data provide an explanation for the experimentally observed influence of TGFbeta on scarring.     

Insulin enhances the growth of cartilage in organ and tissue cultures of mouse neonatal mandibular condyle

Summary Condylar cartilages were cultured in the form of organ cultures on top of collagen sponges in medium containing 2% fetal calf serum and were treated with 3.5–350 nM insulin for 6 days. Doses of 175 nM of insulin caused a marked increase (+96%) in DNA synthesis and in proteoglycan production (+74%), features that manifested themselves structurally by a 60% increase in overall size of the cultured explants. Using a tissue culture system comprised of cartilage progenitor cells, insulin was found to enhance the differentiation of the progenitor cells so that by 6 days in culture and appreciable nodule of differentiated chondrocytes developed. The latter was surrounded by perichondrial cells whereas the extracellular matrix within the newly formed, insulin-induced, nodule reacted positively for cartilagespecific antigens (type II collagen and bone sialoprotein). It is suggested that insulin induces a direct stimulatory effect on progenitor cell proliferation, cartilage differentiation, and extracellular matrix deposition.     

023 Development of Tissue Engineering Skin Based on Acellular Dermal Matrix

Aim: In tissue engineering of the skin, the selection of a scaffold or a matrix in which a cultured cells grow is quite important. We have developed a tissue engineering skin composed of human keratinocytes and fibroblasts on an acellular allogenic dermal matrix (ADM), derived from cryopreserved human skin. Methods: ADM was prepared from cryopreserved split‐thickness human skin by treating with Dispase and Triton X‐100. The tissue engineering skin were produced by seeding human keratinocytes and fibroblasts on ADM. Several days after seeding, the tissue engineering skin was exposed to an air‐liquid interface for another 7 days. Then, the histological structure of the skin and the production of growth factors by the skin were investigated. Results: The produced ADM was found to be completely acellular with remaining structure of the basement membrane components, such as type IV collagen and laminin. The developed tissue engineering skin had stratified keratinocytes on the surface of the ADM migrating fibroblasts in the dermal collagen structure, resembling to the normal skin appearance. It was found that several important growth factors in wound healing process, such as TGF‐α, TGF‐β and VEGF, were produced by the tissue engineering skin. Conclusions: It was suggested that ADM is suitable for a scaffold in tissue engineering of the skin.     

Porcine small intestinal submucosa (SIS): a bioscaffold supporting in vitro primary human epidermal cell differentiation and synthesis of basement membrane proteins

The growth pattern of human epidermal cells, fibroblasts or Swiss mouse 3T3/J2 fibroblasts cultured upon the extracellular matrix (ECM) derived from small intestinal submucosa (SIS) was evaluated. The cell/SIS composites were grown submerged, then maintained in air/liquid interface for 2, 7, 10 or 14 days. The presence of differentiation-related keratins 10, 14 and 16, FN, laminin, collagen type VII and collagen type IV was determined by immunohistochemical methods in SIS alone and in the SIS/cell composite. Only FN could be detected in SIS alone. SIS supported the formation of an epithelial structure with suprabasal expression of K16 and regional suprabasal expression of K10. The epidermal cells were K14 positive and tended to 'invade' the SIS to various degrees. Following the growth of epidermal cells and fibroblasts on the SIS substratum, immunolabeling of FN, laminin, collagen type VII and collagen type IV was observed in a cell-associated pattern. The fibroblasts commonly invaded the SIS, when co-cultivated with epidermal cells on the opposite side of the SIS. The ability of SIS to support epidermal cell/fibroblast attachment, migration and/or proliferation and differentiation with deposition of basement membrane (BM) components indicates that the composite model may be useful for studying cell-matrix interactions and for investigation as a dermal substitute.     

以活性复合真皮基质为载体构建组织工程皮肤的研究

目的 构建含活性真皮基质的组织工程皮肤. 方法将人成纤维细胞(Fb)与Ⅰ型牛胶原混合接种于猪脱细胞真皮基质(PADM)的表面,构建活性真皮替代物.其上接种人表皮细胞进行气-液面培养,获得组织工程皮肤,进行组织学观察. 结果 Fb在胶原内结构完整,与PADM形成复合真皮基质.所构建的组织工程皮肤表皮层结构与人正常皮肤相似,具备基底层、棘层、颗粒层和角质层,细胞之间有桥粒连接,细胞分化良好. 结论 Fb-胶原-PADM真皮替代物可作为较好的构建组织工程皮肤的真皮支架.     

毛乳头细胞诱导毛囊形成的研究

目的 探讨培养的毛乳头细胞在体内外条件下诱导毛囊形成的可能性。方法 采用酶消化法获得毛乳头细胞、真皮鞘细胞、毛囊上、下段及球部细胞，进行毛囊组织工程重建，或用游离细胞混合移植于棵鼠，组织学观察毛囊形成情况。结果 毛囊间表皮细胞、毛囊上段上皮细胞、下段上皮细胞和球部细胞在间质细胞凝胶上均可形成双层结构的组织工程皮肤，在真皮鞘细胞胶原凝胶上毛囊的上、下段上皮细胞形成了毛囊结构，移植于棵鼠后8周毛乳头细胞胶原凝胶诱导毛囊上、下段细胞形成了毛囊。低代毛乳头细胞与毛囊上皮细胞混合移植形成了数量较多、结构典型的毛囊，并有肉眼可见的毛发纤维产生。结论 毛囊的真皮成分细胞即毛乳头细胞、真皮鞘细胞在体内、外均具有诱导毛囊形成的能力，通过与毛囊上皮细胞之间的相互作用，可诱导毛囊形成。     

Epidermal growth factor and insulin act synergistically during diabetic healing

Increased wound collagen catabolism is among the defects of diabetic wound repair. We studied the interactions of topically applied insulin and epidermal growth factor (EGF) in diabetic rats. Polytetrafluoroethylene cylinders were implanted in 80 diabetic rats and removed on postoperative days 1, 5, 10, and 15. Cylinders were analyzed for collagen concentration and collagenase activity. The EGF and insulin promoted a 202% increase over controls in collagen synthesis by day 15, while diabetic rats that received EGF or insulin alone had significantly less collagen than controls. All groups that received insulin had lower collagenase activity than both controls and diabetic rats that received EGF. The individual effects of insulin and EGF added synergistically for a net gain in wound collagen content after 15 days. This gain was not observed with either EGF or insulin alone.