Fibronectin-hyaluronic acid composite hydrogels for three-dimensional endothelial cell culture

Biomaterials that actively promote both wound healing and angiogenesis are of critical importance for many biomedical applications, including tissue engineering. In particular, hyaluronic acid (HA) is an important player that has multiple roles throughout the angiogenic process in the body. Previously, our laboratory has developed photocrosslinkable HA-based scaffolds that promote angiogenesis when implanted in vivo. This paper reports the incorporation of a photocrosslinkable fibronectin (FN) conjugate into three-dimensional (3-D) HA hydrogel networks to enhance endothelial cell adhesion and angiogenesis. The results demonstrate significantly better retention of FN that was photocrosslinked within HA hydrogels compared to FN that was physically adsorbed within HA hydrogels. Increased viability of endothelial cells cultured in 3-D HA hydrogels with photoimmobilized FN, compared to adsorbed FN, was also observed. Endothelial cells were cultured within hydrogels for up to 6 days, a period over which cell proliferation, migration and an angiogenic phenotype were influenced by varying the concentration of incorporated FN. The results demonstrate the potential of these composite hydrogels as biomaterial scaffolds capable of promoting wound healing and angiogenesis.     

Bioactive interpenetrating polymer network hydrogels that support corneal epithelial wound healing

The development and characterization of collagen-coupled poly(ethylene glycol)/poly(acrylic acid) (PEG/PAA) interpenetrating polymer network hydrogels is described. Quantitative amino acid analysis and FITC-labeling of collagen were used to determine the amount and distribution of collagen on the surface of the hydrogels. The bioactivity of the coupled collagen was detected by a conformation-specific antibody and was found to vary with the concentration of collagen reacted to the photochemically functionalized hydrogel surfaces. A wound healing assay based on an organ culture model demonstrated that this bioactive surface supports epithelial wound closure over the hydrogel but at a decreased rate relative to sham wounds. Implantation of the hydrogel into the corneas of live rabbits demonstrated that epithelial cell migration is supported by the material, although the rate of migration and morphology of the epithelium were not normal. The results from the study will be used as a guide toward the optimization of bioactive hydrogels with promise in corneal implant applications such as a corneal onlay and an artificial cornea.     

Effects of fibroblasts and basic fibroblast growth factor on facilitation of dermal wound healing by type I collagen matrices

Healing of large open dermal wounds is associated with decreased values of the tensile strength even up to 6 months post-wounding. Results of previous studies have shown that healing is facilitated in the presence of a type I collagen sponge by promoting deposition of newly synthesized large-diameter collagen fibers parallel to the fibers of the sponge. In this study healing is evaluated in dermal wounds treated with a collagen sponge seeded with fibroblasts or coated with basic fibroblast growth factor (bFGF). Experimental results indicate that the presence of a collagen sponge results in increased wound tensile strength and increased collagen fiber diameters in the upper dermis 15 days post-wounding in an excisional guinea pig dermal wound model. In comparison, dermal wounds treated with collagen sponges seeded with fibroblasts or coated with bFGF showed increased tensile strengths 15 days postimplantation and increased degree of reepithelialization. These results indicate that fibroblast seeding and bFGF coating in conjunction with a type I collagen sponge matrix facilitate early dermal and epidermal wound healing.     

Expansion and delivery of human fibroblasts on micronized acellular dermal matrix for skin regeneration

In order to obtain an abundant supply of autologous dermal fibroblasts for the manufacture of engineered autologous dermal substitutes, we fabricated the micronized acellular dermal matrix (MADM) microcarriers and expanded human fibroblasts on them. This novel approach eliminated the need for the repeated trypsinizations that may disrupt cell–extracellular matrix interactions and impair cell viability. This cell expansion protocol simultaneously formed an engineered particulate dermal substitute (EPDS) avoiding cell reseeded on the scaffolds process. We further tested its feasibility and effectiveness in athymic murine subcutaneous injection and full-thickness cutaneous wound model. Our results showed that MADM microcarriers retained the ultrastructure of the acellular dermal matrix, had good biocompatibility, and supported human fibroblast expansion either as a direct culture substrate or through culturing cells in conditioned medium prepared from them. In the animal study, EPDS formed a thick layer of tissue below the subcutaneous muscle tissue at 3 weeks following EPDS injection into subcutaneous tissue. In full-thickness cutaneous wound, the degree of wound healing with EPDS implantation was better than that without EPDS although healing rates were not significantly different between wounds implanted with or without EPDS. This demonstrates the potential utility of MADM not only as a cell culture substrate to expand fibroblasts but also as a cell transplantation vehicle for skin regeneration, with several advantages over current expansion–transplantation protocols for skin regeneration. In addition, EPDS may be used for cosmetic or reconstructive soft tissue augmentation in a minimally invasive fashion.     

Glycosaminoglycan hydrogel films as bio-interactive dressings for wound healing

Chemically-crosslinked glycosaminoglycan (GAG) hydrogel films were prepared and evaluated as bio-interactive wound dressings. Hyaluronan (HA) and chondroitin sulfate (CS) were first converted to the adipic dihydrazide derivatives and then crosslinked with poly(ethylene glycol) propiondialdehyde to give a polymer network. The crosslinking occurred at neutral pH in minutes at room temperature to give clear, soft hydrogels. After gelation, a solvent-casting method was used to obtain a GAG hydrogel film. A mouse model was used to evaluate the efficacy of these GAG films in facilitating wound healing. Full-thickness wounds were created on the dorsal side of Balb/c mice and were dressed with a GAG film plus Tegaderm' or TegadermT' alone. A significant increase in re-epithelialization was observed on day 5 (p < 0.001) and day 7 (p < 0.05) for wounds treated with a GAG film plus Tegaderm versus those treated with Tegaderm alone. While no significant differences in wound contraction or inflammatory response were found, wounds treated with either HA or CS films showed more fibro-vascular tissue by day 10. The GAG hydrogel films provide a highly hydrated, peri-cellular environment in which assembly of other matrix components. presentation of growth and differentiation factors, and cell migration can readily occur.     

Doxycycline hydrogels with reversible disulfide crosslinks for dermal wound healing of mustard injuries

Doxycycline hydrogels containing reversible disulfide crosslinks were investigated for a dermal wound healing application. Nitrogen mustard (NM) was used as a surrogate to mimic the vesicant effects of the chemical warfare agent sulfur mustard. An 8-arm-poly(ethylene glycol) (PEG) polymer containing multiple thiol (-SH) groups was crosslinked using hydrogen peroxide (H(2)O(2) hydrogel) or 8-arm-S-thiopyridyl (S-TP hydrogel) to form a hydrogel in situ. Formulation additives (glycerin, PVP and PEG 600) were found to promote dermal hydrogel retention for up to 24 h. Hydrogels demonstrated high mechanical strength and a low degree of swelling (< 1.5%). Doxycycline release from the hydrogels was biphasic and sustained for up to 10-days in vitro. Doxycycline (8.5 mg/cm(3)) permeability through NM-exposed skin was elevated as compared to non vesicant-treated controls at 24, 72 and 168 h post-exposure with peak permeability at 72 h. The decrease in doxycycline permeability at 168 h correlates to epidermal re-epithelialization and wound healing. Histology studies of skin showed that doxycycline loaded (0.25% w/v) hydrogels provided improved wound healing response on NM-exposed skin as compared to untreated skin and skin treated with placebo hydrogels in an SKH-1 mouse model. In conclusion, PEG-based doxycycline hydrogels are promising for dermal wound healing application of mustard injuries.     

纳米银－猪脱细胞真皮基质敷料治疗浅Ⅱ度烧伤创面的临床观察

目的观察纳米银－猪脱细胞真皮基质敷料在临床上治疗浅Ⅱ度烧伤创面的临床疗效。 方法2014年1月到2015年12月,选取北京军区总医院烧伤整形科收治的浅Ⅱ度烧伤患者90例,按入院顺序依次编码,采用随机排列数字表法将90例患者分为纳米银敷料组、猪脱细胞真皮基质敷料组及纳米银－猪脱细胞真皮基质敷料组,每组30例。患者入院当天,拍照计算创面面积,并用咽拭子取创面分泌物作细菌培养,行创面清创术,分别在创面上敷以纳米银敷料、猪脱细胞真皮基质敷料以及纳米银－猪脱细胞真皮基质敷料。于治疗后第5天,用咽拭子取创面分泌物作细菌培养;采用痛觉评分标准,通过询问与观察患者换药时的痛觉情况,评估患者换药时痛觉评分。于治疗后第7天,拍照计算创面面积,计算创面愈合率。记录创面最终愈合时间。数据比较采用单因素方差分析、χ²检验及SNK－q检验。 结果纳米银敷料组、猪脱细胞真皮基质敷料组及纳米银－猪脱细胞真皮基质敷料组在治疗后第5天创面细菌培养阳性数结果分别为2例(6.6%)、9例(30.0%)、1例(3.3%),3组结果比较,差异有统计学意义(χ²＝10.962,P＝0.004);纳米银－猪脱细胞真皮基质敷料组的细菌培养阳性数结果显著优于猪脱细胞真皮基质敷料组,差异有统计学意义(χ²＝7.680,P＝0.006)。纳米银敷料组、猪脱细胞真皮基质敷料组及纳米银－猪脱细胞真皮基质敷料组治疗后第5天的痛觉评分[(8.6±0.5)、(6.6±0.8)、(0.6±1.3)分],治疗后第7天的创面愈合率[(61.67±18.22)%、(86.77±15.32)%、(99.80±0.56)%],创面愈合时间[(11.5±1.3)、(10.3±0.7)、(7.3±0.7)d],组间差异均有统计学意义差异(F＝201.7、19.9、55.7,P值均小于0.05);纳米银－猪脱细胞真皮基质敷料组治疗后第5天的痛觉评分显著低于其余两组,差异均有统计学意义(P值均小于0.05);治疗后第7天的创面愈合率明显优于其余两组,差异均有统计学意义(P值均小于0.05);创面愈合时间显著短于其余两组,比较差异均有统计学意义(P值均小于0.05)。 结论纳米银－猪脱细胞真皮基质敷料具有抗感染、促进创面愈合及减轻换药痛觉的作用。     

An in vitro examination of an extracellular matrix scaffold for use in wound healing

This paper describes evidence that an extracellular matrix (ECM) secreted by human umbilical vein endothelial cells (HUVECs) assembled on gelatin coated plates overlaid by a mixed matrix secreted by human dermal microvascular endothelial cells (HDMECs) and human dermal fibroblasts provides a viable acellular scaffold for use in wound healing. Trypsinized epidermal keratinocytes or colonies from Dispase-digested fresh and cadaver skin tissue adhered and proliferated on either HUVECs ECM/gelatin or mixed matrix overlaid on HUVECs ECM/gelatin. An epithelial-mesenchymal interaction, previously thought to be tissue-specific, was exposed as well as concomitant integrin versatility. Furthermore, heterologous HDMECs and dermal fibroblasts attached and proliferated on the mixed matrix as well as HUVECs ECM. The conditioned medium from HUVECs (HUVECs CM) was found to neutralize the lingering after effects of Dispase, and could be used for the tissue culture of epidermal keratinocytes, HDMECs and dermal fibroblasts, which share related extracellular secretions. Taken together, these results indicate that cultured epithelial autografts can be redesigned to include both epithelial and dermal elements, and advances the acellular 'sandwich' ECM scaffold as a possible structural replacement for the lamina densa and lamina lucida, damaged or completely missing in some wounds and burns.     

Skin tissue engineering

The major applications of tissue-engineered skin substitutes are in promoting the healing of acute and chronic wounds. Several approaches have been taken by commercial companies to develop products to address these conditions. Skin substitutes include both acellular and cellular devices. While acellular skin substitutes act as a template for dermal formation, this discussion mainly covers cellular devices. In addressing therapeutic applications in tissue engineering generally, a valuable precursor is an understanding of the mechanism of the underlying pathology. While this is straightforward in many cases, it has not been available for wound healing. Investigation of the mode of action of the tissue-engineered skin substitutes has led to considerable insight into the mechanism of formation, maintenance and treatment of chronic wounds. Four aspects mediating healing are considered here for their mechanism of action: (i) colonization of the wound bed by live fibroblasts in the implant, (ii) the secretion of growth factors, (iii) provision of a suitable substrate for cell migration, particularly keratinocytes and immune cells, and (iv) modification of the immune system by secretion of neutrophil recruiting chemokines. An early event in acute wound healing is an influx of neutrophils that destroy planktonic bacteria. However, if the bacteria are able to form biofilm, they become resistant to neutrophil action and prevent reepithelialization. In this situation the wound becomes chronic. In chronic wounds, fibroblasts show a senescence-like phenotype with decreased secretion of neutrophil chemoattractants that make it more likely that biofilms become established. Treatment of the chronic wounds involves debridement to eliminate biofilm, and the use of antimicrobials. A role of skin substitutes is to provide non-senescent fibroblasts that attract and activate neutrophils to prevent biofilm re-establishment. The emphasis of the conclusion is the importance of preventing contaminating bacteria becoming established and forming biofilms.     

Evaluation of an in situ forming hydrogel wound dressing based on oxidized alginate and gelatin

Wound dressings that can be formed in situ offer several advantages over the use of preformed dressings such as conformability without wrinkling or fluting in the wound bed, ease of application and improved patient compliance and comfort. Here we describe such an in situ forming hydrogel wound dressing from gelatin, oxidized alginate and borax. Periodate oxidized alginate rapidly cross-links proteins such as gelatin in the presence of borax to give in situ forming hydrogels that are both non-toxic and biodegradable. The composite matrix has the haemostatic effect of gelatin, the wound healing-promoting feature of alginate and the antiseptic property of borax to make it a potential wound dressing material. The hydrogel was found to have a fluid uptake of 90% of its weight which would prevent the wound bed from accumulation of exudates. The water vapour transmission rate (WVTR) of the hydrogel was found to be 2686+/-124 g/m2/day indicating that the hydrogel can maintain a moist environment over wound bed in moderate to heavily exuding wound which would enhance epithelial cell migration during the healing process. The wound healing efficacy of hydrogel was evaluated in experimental full thickness wounds using a rat model which demonstrated that within 2 weeks, the wound covered with gel was completely filled with new epithelium without any significant adverse reactions. These in situ forming hydrogels fulfil many critical elements desirable in a wound dressing material.     

Biological characterization of EDC-crosslinked collagen-hyaluronic acid matrix in dermal tissue restoration

Porous collagen matrices crosslinked with various amounts of hyaluronic acid (HA) by 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) were developed as scaffolds for dermal tissue regeneration. The effect of HA on cells in accordance with HA concentrations in the collagenous matrices was investigated using cultures of fetal human dermal fibroblasts, and the effect of EDC-crosslinked collagen-HA matrix on wound size reduction was also evaluated in vivo. Scanning electron microscopic views of the matrices demonstrated that all of the collagen-HA matrices had interconnected pores with mean diameters of 150-250 microm. An HA matrix retention test showed that the concentration of HA decreased slowly after an initial rapid decrease over 24h. Fetal human dermal fibroblasts adhered well to all of the collagen-based matrices as compared with the Porous polyurethane matrix used as a control. An 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide based proliferation test and the hematoxylin and eosin staining of a 2 week cultured matrix showed that the proliferation of fibroblasts was enhanced on a 9.6% HA contained collagen matrix. No significant difference was in terms of fibroblast migration into the various types of scaffolds as HA content was increased. In vivo testing showed that dermis treated with collagen or collagen-HA matrix was thicker than the control, and epithelial regeneration was accelerated, and collagen synthesis increased. However, no significant effect of HA on wound size reduction was found.     

Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor

Replacement of wounded skin requires the initially florid cellular response to abate and even regress as the dermal layer returns to a relatively paucicellular state. The signals that direct this "stop and return" process have yet to be deciphered. CXCR3 chemokine receptor and its ligand CXCL11/IP-9/I-TAC are expressed by basal keratinocytes and CXCL10/IP-10 by keratinocytes and endothelial cells during wound healing in mice and humans. In vitro, these ligands limit motility in dermal fibroblasts and endothelial cells. To examine whether this signaling pathway contributes to wound healing in vivo, full-thickness excisional wounds were created on CXCR3 wild-type (+/+) or knockout (-/-) mice. Even at 90 days, long after wound closure, wounds in the CXCR3(-/-) mice remained hypercellular and presented immature matrix components. The CXCR3(-/-) mice also presented poor remodeling and reorganization of collagen, which resulted in a weakened healed dermis. This in vivo model substantiates our in vitro findings that CXCR3 signaling is necessary for inhibition of fibroblast and endothelial cell migration and subsequent redifferentiation of the fibroblasts to a contractile state. These studies establish a pathophysiologic role for CXCR3 and its ligand during wound repair.     

异种（猪）去细胞真皮基质修复深度烧伤创面的临床应用

目的观察异种（猪）去细胞真皮基质修复深度烧伤创面的临床效果。方法临床选取40例深度烧伤患者，同一患者不同创面分别使用异种（猪）去细胞真皮基质＋自体刃厚皮（复合皮组）和自体刃厚皮（对照组）覆盖切痂创面，术后定期行创面愈合率和收缩率比较，同时行组织学观察。结果复合皮组创面愈合良好，两组创面愈合率比较无统计学差异；但复合皮组皮肤弹性较好，收缩率显著低于对照组；组织学观察复合皮组真皮胶原纤维排列有序，基底膜结构完整。结论异种（猪）去细胞真皮基质与自体刃厚皮复合移植于深度烧伤切痂创面，可改善创面愈合质量。     

Mechanically strong double network photocrosslinked hydrogels from N,N-dimethylacrylamide and glycidyl methacrylated hyaluronan

Hyaluronan (HA) is a natural polysaccharide abundant in biological tissues and it can be modified to prepare biomaterials. In this work, HA modified with glycidyl methacrylate was photocrosslinked to form the first network (PHA), and then a series of highly porous PHA/N,N-dimethylacrylamide (DAAm) hydrogels (PHA/DAAm) with high mechanical strength were obtained by incorporating a second network of photocrosslinked DAAm into PHA network. Due to the synergistic effect produced by double network (DN) structure, despite containing 90% of water, the resulting PHA/DAAm hydrogel showed a compressive modulus and a fracture stress over 0.5 MPa and 5.2 MPa, respectively. Compared to the photocrosslinked hyaluronan single network hydrogel, which is generally very brittle and fractures easily, the PHA/DAAm hydrogels are ductile. Mouse dermal fibroblast was used as a model cell line to validate in vitro non-cytotoxicity of the PHA/DAAm hydrogels. Cells deposited extracellular matrix on the surface of these hydrogels and this was confirmed by positive staining of Type I collagen by Sirius Red. The PHA/DAAm hydrogels were also resistant to biodegradation and largely retained their excellent mechanical properties even after 2 months of co-culturing with fibroblasts.     

Stimulation of skin repair is dependent on fibroblast source and presence of extracellular matrix

In this study in vitro and in vivo functions were compared between cultured dermal equivalents produced with human fibroblasts isolated either from papillary dermis or adipose tissue of the same donors. Papillary dermal fibroblasts had a normal spindle cell shape; in contrast, adipose tissue fibroblasts had a stellate cell shape, actin stress fibers containing alpha-smooth muscle actin, multiple narrow extensions at their edges, and longer focal adhesion plaques. After dynamic culture for 14 days in PEGT/PBT carrier scaffolds, cell numbers between the two cell sources were comparable, but tissue morphology was different between the cultured groups. In addition, papillary fibroblasts had deposited significantly more glycosaminoglycans (214 +/- 15 versus 159 +/- 21 microg, p < 0.001) and a lower amount of collagen (49 +/- 14 versus 111 +/- 25 microg of hydroxyproline, p < 0.001) than had adipose fibroblasts. Moreover, the latter constructs were significantly more contracted than the papillary fibroblast-cultured constructs (78 +/- 6 versus 96 +/- 3%, p < 0.001). In comparison with the influence of cultured dermal equivalents on wound healing, the transplantation of five groups (control acellular carrier, papillary fibroblast-seeded construct, adipose fibroblast-seeded construct, papillary fibroblast-cultured construct, and adipose fibroblast-cultured construct) to full-thickness wounds on the backs of athymic mice showed clear differences in angiogenesis and tissue ingrowth after 10 days, and in reepithelialization after 21 days. After 10 days, the level of vascular ingrowth in the carrier (von Willebrand staining) for the five groups was as follows: adipose fibroblast-cultured > papillary fibroblast-cultured = adipose fibroblast-seeded > papillary fibroblast-seeded > acellular carrier. After 21 days, only the acellular carriers were not vascularized and the papillary fibroblast-seeded constructs were not completely vascularized. Complete wound reepithelialization (92 +/- 12%) was observed only in the group treated with adipose cultured constructs. Wound contraction was not observed. Staining for HLA-ABC and alpha-smooth muscle actin showed that human fibroblasts had survived and that adipose fibroblasts continued to express the actin isoform. These results showed not only stimulation of skin repair when fibroblasts were present in the carrier, but also significant positive effects of the deposited extracellular matrix (ECM) in the carrier. In addition, the adipose fibroblast-seeded construct, and especially the adipose fibroblast-cultured construct, significantly stimulated angiogenesis and reepithelialization when compared with their corresponding papillary fibroblast constructs. Apparently, tissue source or fibroblast phenotype and the presence of ECM play a crucial role in the stimulation of (impaired) healing and engineering of dermal equivalents.     

Synthesis and properties of waterborne polyurethane hydrogels for wound healing dressings

To accomplish ideal wound healing dressing, a series of waterborne polyurethane (WBPU) hydrogels based on polyethylene glycol (PEG) were synthesized by polyaddition reaction in an emulsion system. The stable WBPU hydrogels which have remaining weight of above 85% were obtained. The effect of the soft segment (PEG) content on water absorbability of WBPU hydrogels was investigated. Water absorption % and equilibrium water content (%) of the WBPU hydrogel significantly increased in proportion to PEG content and the time of water-immersion. The maximum water absorption % and equilibrium water content (%) of WBPU hydrogels containing various PEG contents were in the range of 409-810% and 85-96%, respectively. The water vapor transmission rate of the WBPU hydrogels was found to be in the range of 1490-3118 g/m(2)/day. These results suggest that the WBPU hydrogels prepared in this study may have high potential as new wound dressing materials, which provide and maintain the adequate moist environment required to prevent scab formation and dehydration of the wound bed. By the wound healing evaluation using full-thickness rat model experiment, it was found that the wound covered with a typical WBPU hydrogel (HG-78 sample) was completely filled with new epithelium without any significant adverse reactions.     

Comparative study on motility of the cultured fetal and neonatal dermal fibroblasts in extracellular matrix.

One of the differences between fetal and adult skin healing is the ability of fetal wounds heal without contraction and scar formation. Extracellular matrix (ECM) provides a substratum for cells adhesion, migration, and proliferation and can directly influence the form and function of cells. As motility is essential for many important biological events, including wound healing, inflammatory response, embryonic development, and tumor metastasis, this study was designed to compare the motilities cultured dermal fetal and neonatal fibroblasts in the extracellular matrix. The motility of cultured fetal and neonatal fibroblasts was compared using a video-microscopy system that was developed in combination with a self-designed CO2 mini-incubator. To determine migration speed, cells were viewed with a 4X phase-contrast lens and video recorded. Images were captured using a color CCD camera and saved in 8-bit full-color mode. We found that cultured fetal fibroblasts move faster than neonatal fibroblast on type I collagen (fetal fibroblast, 15.1 micrometer/hr; neonatal fibroblast, 13.7 micrometer/hr), and in fibronectin (fetal fibroblast, 13.2 micrometer/hr; neonatal fibroblast, 13.0 micrometer/hr) and hyaluronic acid (fetal fibroblast, 11 micrometer/hr; neonatal fibroblast, 9.8 micrometer/hr).     

Altered dermal fibroblast behavior in a collagen V haploinsufficient murine model of classic Ehlers–Danlos syndrome

Mutations in collagen V are associated with classic Ehlers–Danlos syndrome (EDS). A significant percentage of these mutations result in haploinsufficiency for collagen V. The purpose of this work was to determine if changes in collagen V expression are associated with altered dermal fibroblast behavior contributing to the poor wound healing response. A haploinsufficient Col5a1^+/? mouse model of EDS was utilized. In vivo wound healing studies demonstrated that mutant mice healed significantly slower than Col5a1^+/+ mice. The basis for this difference was examined in vitro using dermal fibroblast strains isolated from Col5a1^+/? and Col5a1^+/+ mice. Fibroblast proliferation was determined for each strain by counting cells at different time points after seeding as well as using the proliferation marker Ki-67. Fibroblast attachment to collagens I and III and fibronectin also was analyzed. In addition, in vitro scratch wounds were used to analyze fibroblast wound closure. Significantly decreased fibroblast proliferation was observed in Col5a1^+/? compared to Col5a1^+/+ fibroblasts. Our data indicate that the decreased fibroblast number was not due to apoptosis. Wildtype Col5a1^+/+ fibroblasts attached significantly better to components of the wound matrix (collagens I and III and fibronectin) than Col5a1^+/? fibroblasts. A significant difference in in vitro scratch wound closure rates also was observed. Col5a1^+/+ fibroblasts closed wounds in 22 h, while Col5a1^+/? fibroblasts demonstrated ~80% closure. There were significant differences in closure at all time points analyzed. Our data suggest that decreased fibroblast proliferation, extracellular matrix attachment, and migration contribute to the decreased wound healing response in classic EDS.     

FGF-1 and proteolytically mediated cleavage site presentation influence three-dimensional fibroblast invasion in biomimetic PEGDA hydrogels

Controlled scaffold degradation is a critical design criterion for the clinical success of tissue-engineered constructs. Here, we exploited a biomimetic poly(ethylene glycol) diacrylate (PEGDA) hydrogel system immobilized with tethered YRGDS as the cell adhesion ligand and with either single (SSite) or multiple (MSite) collagenase-sensitive domains between crosslinks, to systematically study the effect of proteolytic cleavage site presentation on hydrogel degradation rate and three-dimensional (3-D) fibroblast invasion in vitro. Through the incorporation of multiple collagenase-sensitive domains between cross-links, hydrogel degradation rate was controlled and enhanced independent of alterations in compressive modulus. As compared to SSite hydrogels, MSite hydrogels resulted in increased 3-D fibroblast invasion in vitro, which occurred over a wider range of compressive moduli. Furthermore, encapsulated soluble acidic fibroblast growth factor (FGF-1), a potent mitogen during processes such as vascularization and wound healing, was incorporated into SSite and MSite PEGDA scaffolds to determine its in vitro potential on fibroblast cell invasion. Hydrogels containing soluble FGF-1 significantly enhanced 3-D fibroblast invasion in a dose-dependent manner within the different types of PEG matrices investigated over a period of 15 days. The methodology presented provides flexibility in designing PEG scaffolds with desired mechanical properties, but with increased susceptibility to proteolytically mediated degradation. These results indicate that effective tuning of initial matrix stiffness and hydrogel degradation kinetics plays a critical role in effectively designing PEG scaffolds that promote controlled 3-D cellular behavior and in situ tissue regeneration.