Microcirculation surrounding end-stage human chronic skin wounds is associated with endoglin/CD146/ALK-1 expression, endothelial cell proliferation and an absence of p16Ink4a.

Angiogenesis is an essential part of normal skin healing, re-establishing blood flow in developing granulation tissue. Non-healing skin wounds are associated with impaired angiogenesis and although the role of re-establishing macroscopic blood flow to limbs to prevent wound chronicity is well investigated, less is known about vascular alterations at the microcirculatory level. We hypothesised that significant phenotypic changes would be evident in blood vessels surrounding chronic skin wounds. Wound edge tissue, proximal to wound (2 cm from wound edge) and non-involved skin (>10 cm from wound edge) was harvested under informed consent from 20 patients undergoing elective amputation due to critical limb ischemia. To assess blood vessel structure and viability, tissue was prepared for histological analysis and labelled with antibodies specific for PECAM-1 (CD31), CD146, endoglin, ALK-1, ALK-5, and p16Ink4a as a marker of cellular senescence. Density of microvasculature was significantly increased in wound edge dermis, which was concomitant with increased labelling for endoglin and CD146. The number of CD31 positive vessel density was unchanged in wound edge tissue relative to non-involved tissue. Co-labelling of endoglin with the transforming growth factor receptor ALK-1, and to a lesser extent ALK-5, demonstrated activation of endothelial cells which correlated with PCNA labelling indicative of proliferation. Analysis of p16Ink4a staining showed a complete lack of immunoreactivity in the vasculature and dermis, although staining was evident in sub-populations of keratinocytes. We conclude that the endoglin-ALK-1-endothelial proliferation axis is active in the vasculature at the edge of chronic skin wounds and is not associated with p16Ink4a mediated senescence. This information could be further used to guide treatment of chronic skin wounds and optimise debridement protocols.

Screening of functionalized collagen membranes with a porcine periodontal regeneration model.

OBJECTIVES: Current methods for periodontal regeneration do not promote collagen fiber insertions into new bone and cementum. We used a pig wound model to screen different functionalized collagen membranes in promoting periodontal reattachment to root surfaces. METHODS: Treatment groups included (1) control with no membranes, (2) collagen-coated membranes, (3) membranes with insulin-like growth factor-1 (IGF-1), (4) membranes with amelotin, or (5) membranes attached with calcium phosphate cement (CPC), or with CPC combined with IGF-1. Flap procedures were performed on mandibular and maxillary premolars of each pig. RESULTS: Histomorphometric, micro-CT, and clinical measurements obtained at 4 and 12 weeks after surgery showed cementum formation on denuded roots and reformation of alveolar bone, indicating that the pig model can model healing responses in periodontal regeneration. Calcium phosphate cement simplified procedures by eliminating the need for sutures and improved regeneration of alveolar bone (p < 0.05) compared with other treatments. There was a reduction (p < 0.05) of PD only for the IGF group. Large observed variances between treatment groups indicated that a priori power analyses should be conducted to optimize statistical analysis. CONCLUSIONS: Pigs can model discrete elements of periodontal healing using collagen-based, functionalized membranes. Screening indicates that membrane anchorage with calcium phosphate cements improve regeneration of alveolar bone.

The pig as a model system for investigating the recruitment and contribution of myofibroblasts in skin healing.

In the skin-healing field, porcine models are regarded as a useful analogue for human skin due to their numerous anatomical and physiological similarities. Despite the widespread use of porcine models in skin healing studies, the initial origin, recruitment and transition of fibroblasts to matrix-secreting contractile myofibroblasts are not well defined for this model. In this review, we discuss the merit of the pig as an animal for studying myofibroblast origin, as well as the challenges associated with assessing their contributions to skin healing. Although a variety of wound types (incisional, partial thickness, full thickness, burns) have been investigated in pigs in attempts to mimic diverse injuries in humans, direct comparison of human healing profiles with regards to myofibroblasts shows evident differences. Following injury in porcine models, which often employ juvenile animals, myofibroblasts are described in the developing granulation tissue at 4 days, peaking at Days 7-14, and persisting at 60 days post-wounding, although variations are evident depending on the specific pig breed. In human wounds, the presence of myofibroblasts is variable and does not correlate with the age of the wound or clinical contraction. Our comparison of porcine myofibroblast-mediated healing processes with those in humans suggests that further validation of the pig model is essential. Moreover, we identify several limitations evident in experimental design that need to be better controlled, and standardisation of methodologies would be beneficial for the comparison and interpretation of results. In particular, we discuss anatomical location of the wounds, their size and depth, as well as the healing microenvironment (wet vs. moist vs. dry) in pigs and how this could influence myofibroblast recruitment. In summary, although a widespread model used in the skin healing field, further research is required to validate pigs as a useful analogue for human healing with regards to myofibroblasts.

Wound healing and fibrosis: a contrasting role for periostin in skin and the oral mucosa.

Both skin and oral mucosa are characterized by the presence of keratinized epithelium in direct apposition to an underlying collagen-dense connective tissue. Despite significant overlap in structure and physiological function, skin and the oral mucosa exhibit significantly different healing profiles in response to injury. The oral mucosa has a propensity for rapid restoration of barrier function with minimal underlying fibrosis, but in contrast, skin is associated with slower healing and scar formation. Modulators of cell function, matricellular proteins have been shown to play significant roles in cutaneous healing, but their role in restoration of the oral mucosa is poorly defined. As will be discussed in this review, over the last 12 years our research group has been actively investigating the role of the profibrotic matricellular protein periostin in tissue homeostasis and fibrosis, as well as healing, in both skin and gingiva. In the skin, periostin is highly expressed in fibrotic scars and is upregulated during cutaneous wound repair, where it facilitates myofibroblast differentiation. In contrast, in gingival healing, periostin regulates extracellular matrix synthesis but does not appear to be associated with the transition of mesenchymal cells to a contractile phenotype. The significance of these findings will be discussed, with a focus on periostin as a potential therapeutic to augment healing of soft tissues or suppress fibrosis.

JNK Signaling as a Key Modulator of Soft Connective Tissue Physiology, Pathology, and Healing.

In healthy individuals, the healing of soft tissues such as skin after pathological insult or post injury follows a relatively predictable and defined series of cell and molecular processes to restore tissue architecture and function(s). Healing progresses through the phases of hemostasis, inflammation, proliferation, remodeling, and concomitant with re-epithelialization restores barrier function. Soft tissue healing is achieved through the spatiotemporal interplay of multiple different cell types including neutrophils, monocytes/macrophages, fibroblasts, endothelial cells/pericytes, and keratinocytes. Expressed in most cell types, c-Jun N-terminal kinases (JNK) are signaling molecules associated with the regulation of several cellular processes involved in soft tissue wound healing and in response to cellular stress. A member of the mitogen-activated protein kinase family (MAPK), JNKs have been implicated in the regulation of inflammatory cell phenotype, as well as fibroblast, stem/progenitor cell, and epithelial cell biology. In this review, we discuss our understanding of JNKs in the regulation of cell behaviors related to tissue injury, pathology, and wound healing of soft tissues. Using models as diverse as Drosophila, mice, rats, as well as human tissues, research is now defining important, but sometimes conflicting roles for JNKs in the regulation of multiple molecular processes in multiple different cell types central to wound healing processes. In this review, we focus specifically on the role of JNKs in the regulation of cell behavior in the healing of skin, cornea, tendon, gingiva, and dental pulp tissues. We conclude that while parallels can be drawn between some JNK activities and the control of cell behavior in healing, the roles of JNK can also be very specific modes of action depending on the tissue and the phase of healing.

Periostin as a multifunctional modulator of the wound healing response.

During tissue healing, the dynamic and temporal alterations required for effective repair occur in the structure and composition of the extracellular matrix (ECM). Matricellular proteins (MPs) are a group of diverse non-structural ECM components that bind cell surface receptors mediating interactions between the cell and its microenviroment, effectively regulating adhesion, migration, proliferation, signaling, and cell phenotype. Periostin (Postn), a pro-fibrogenic secreted glycoprotein, is defined as an MP based on its expression pattern and regulatory roles during development and healing and in disease processes. Postn consists of a typical signal sequence, an EMI domain responsible for binding to fibronectin, four tandem fasciclin-like domains that are responsible for integrin binding, and a C-terminal region in which multiple splice variants originate. This review focuses specifically on the role of Postn in wound healing and remodeling, an area of intense research during the last 10 years, particularly as related to skin healing and myocardium post-infarction. Postn interacts with cells through various integrin pairs and is an essential downstream effector of transforming growth factor-ß superfamily signaling. Across various tissues, Postn is associated with the pro-fibrogenic process: specifically, the transition of fibroblasts to myofibroblasts, collagen fibrillogenesis, and ECM synthesis. Although the complexity of Postn as a modulator of cell behavior in tissue healing is only beginning to be elucidated, its expression is clearly a defining event in moving wound healing through the proliferative and remodeling phases.

Regulation of matrix remodelling phenotype in gingival fibroblasts by substratum topography.

Gingival connective tissue often has a composition resembling that of scar surrounding dental implant abutments. Increased cell adhesion, α-smooth muscle actin (α-SMA) expression and increased extracellular matrix deposition are a hallmark of fibrotic cells, but how topographic features influence gingival fibroblast adhesion and adoption of the α-SMA positive myofibroblast phenotype associated with scarring is unknown. The purpose of the present study was to demonstrate whether implant topographies that limit adhesion formation would reduce myofibroblast differentiation and extracellular matrix deposition. Human gingival fibroblasts were cultured on PT (smooth) and SLA (roughened) titanium discs for varying time-points. At 1 and 2 weeks after seeding, incorporation of α-SMA into stress-fibre bundles and fibronectin deposition was significantly higher on PT than SLA surfaces indicating differentiation of the cells towards a myofibroblast phenotype. Analysis of adhesion formation demonstrated that cells formed larger adhesions and more stable adhesions on PT, with more nascent adhesions observed on SLA. Gene expression analysis identified up-regulation of 15 genes at 24 hrs on SLA versus PT associated with matrix remodelling. Pharmacological inhibition of Src/FAK signalling in gingival fibroblasts on PT reduced fibronectin deposition and CCN2 expression. We conclude that topographical features that reduce focal adhesion stability could be applied to inhibit myofibroblast differentiation in gingival fibroblasts.

Periostin modulates myofibroblast differentiation during full-thickness cutaneous wound repair.

The matricellular protein periostin is expressed in the skin. Although periostin has been hypothesized to contribute to dermal homeostasis and repair, this has not been directly tested. To assess the contribution of periostin to dermal healing, 6 mm full-thickness excisional wounds were created in the skin of periostin-knockout and wild-type, sex-matched control mice. In wild-type mice, periostin was potently induced 5-7 days after wounding. In the absence of periostin, day 7 wounds showed a significant reduction in myofibroblasts, as visualized by expression of α-smooth muscle actin (α-SMA) within the granulation tissue. Delivery of recombinant human periostin by electrospun collagen scaffolds restored α-SMA expression. Isolated wild-type and knockout dermal fibroblasts did not differ in in vitro assays of adhesion or migration; however, in 3D culture, periostin-knockout fibroblasts showed a significantly reduced ability to contract a collagen matrix, and adopted a dendritic phenotype. Recombinant periostin restored the defects in cell morphology and matrix contraction displayed by periostin-deficient fibroblasts in a manner that was sensitive to a neutralizing anti-ß1-integrin and to the FAK and Src inhibitor PP2. We propose that periostin promotes wound contraction by facilitating myofibroblast differentiation and contraction.

Assessing the fate and contribution of Foxd1-expressing embryonic precursors and their progeny in palatal development, homeostasis and excisional repair.

Oral mucosal tissues heal rapidly with minimal scarring, although palatal mucosa can be associated with excessive fibrosis in response to injury. Investigations on the balance between neovascularization and tissue repair suggests regulation of angiogenesis is an important determinant of repair versus scarring. Associated with pericyte mediated fibrosis in kidney injury, FoxD1 is implicated in growth centres during cranio-facial development, although which cell lineages are derived from these embryonic populations in development and in adult animals is unknown. Using a lineage tracing approach, we assessed the fate of embryonic Foxd1-expressing progenitor cells and their progeny in palatal development and during wound healing in adult mice. During palatal development as well as in post-natal tissues, Foxd1-lineage progeny were associated with the vasculature and the epineurium. Post-injury, de novo expression of FoxD1 was not detectable, although Foxd1-lineage progeny expanded while exhibiting low association with the fibroblast/myofibroblast markers PDGFα, PDGFß, vimentin, α-smooth muscle actin, as well as the neuronal associated markers S100ß and p75NTR. Foxd1-lineage progeny were primarily associated with CD146, CD31, and to a lesser extent CD105, remaining in close proximity to developing neovascular structures. Our findings demonstrate that FoxD1 derived cells are predominantly associated with the palatal vasculature and provide strong evidence that FoxD1 derived cells do not give rise to populations involved directly in the scarring of the palate.

Contact Guidance of Connective Tissue Fibroblasts on Submicrometer Anisotropic Topographical Cues Is Dependent on Tissue of Origin, ß1 Integrins, and Tensin-1 Recruitment.

The substratum topography of both natural and synthetic materials is a prominent regulator of cell behaviors including adhesion, migration, matrix fibrillogenesis, and cell phenotype. Connective tissue fibroblasts are known to respond to repeating groove topographical modifications by aligning and exhibiting directed migration, a phenomenon termed contact guidance. Although both reside in collagen rich connective tissues, dermal and gingival fibroblasts are known to exhibit differences in phenotype during wound healing, with gingival tissue showing a fetal-like scarless response. Differences in adhesion formation and maturation are known to underlie both a scarring phenotype and cell response to topographical features. Utilizing repeating groove substrates with periodicities of 600, 900, and 1200 nm (depth, 100 nm), we investigated the roles of integrins αvß3 and ß1 associated adhesions on contact guidance of human gingival (HGFs) and dermal fibroblasts (HDFs). HGFs showed a higher degree of orientation with the groove long axis than HDFs, with alignment of both vinculin and tensin-1 evident on 600 and 900 nm periodicities in both cell types. Orientation with grooves of any periodicity in HGFs and HDFs did not alter the adhesion number or area compared to smooth control surfaces. Growth of both cell types on all periodicities reduced fibronectin fibrillogenesis compared to control surfaces. Independent inhibition of integrin αvß3 and ß1 in both cell types induced changes in spreading up to 6 h and reduced alignment with the groove long axis. At 24 h post-seeding with blocking antibodies, HGFs recovered orientation, but in HDFs, blocking of ß1, but not αvß3 integrins, inhibited alignment. Blocking of ß1 and αvß3 in HDFs, but not HGFs, inhibited tensin-1-associated fibrillar adhesion formation. Furthermore, inhibition of ß1 integrins in HDFs, but not HGFs, resulted in recruitment of tensin-1 to αvß3 focal adhesions, preventing HDFs from aligning with the groove long axis. Our work demonstrates that tensin-1 localization with specific integrins in adhesion sites is an important determinant of contact guidance. This work emphasizes further the need for tissue-specific biomaterials, when integration into host tissues is required.

Creating 3D angiogenic growth factor gradients in fibrous constructs to guide fast angiogenesis.

Fast angiogenesis in 3D fibrous constructs that mimic the morphology of the extracellular matrix remains challenging due to limited porosity in the densely packed constructs. We investigated whether mimicking the in vivo chemotaxis microenvironment for native blood vessel formation would stimulate angiogenesis in the fibrous constructs. The chemotaxis microenvironment was created by introducing 3D angiogenic growth factor gradients into the constructs. We have developed a technique that can quickly fabricate (∼40 min) such 3D gradients by simultaneously electrospinning polycaprolactone (PCL) fibers, encapsulating gradient amount of bFGF (stabilized by heparin) into poly(lactide-co-glycolide) (PLGA) microspheres, and electrospraying the microspheres into PCL fibers. Gradient formation was confirmed by fluorescence microscopy. Gradients with different steepnesses were obtained by modulating the initial concentration of the bFGF solution. All of the constructs were able to sustainedly release bioactive bFGF over a 28 day period. The release kinetics was dependent on the bFGF loading and steepness of the gradient. In vitro cell migration study demonstrated that bFGF gradients significantly increased the depth of cell migration. To assess the efficacy of bFGF gradients in inducing angiogenesis, we implanted constructs subcutaneously using mouse model. bFGF gradients significantly promoted cell penetration into the constructs. After 10 days of implantation, a high density of mature blood vessels (positive to both CD31 and α-SMA) were formed in the constructs. Vessel density was increased with the increase in steepness of the bFGF gradient. These gradient constructs may have potential to engineer vascularized tissues for various applications.

Initial assessment of gingival biotype as a potential source of variability in the migration, contraction and gene expression of fibroblasts.

OBJECTIVE: Gingival biotype refers to the clinical classification of gingiva based on the thickness of the tissue, with thick gingival tissues more resistant to trauma and recession than the thin variant. However, to date there has never been an analysis of whether fibroblasts isolated from different biotypes possess inherent phenotypic differences. We hypothesized that gingival fibroblasts from thick and thin biotype would exhibit differences in migration, contraction and gene expression in vitro in the presence of either transforming growth factor beta one (TGF-ß1) or tumor necrosis factor alpha (TNFα), two major cytokines involved in wound repair. DESIGN: Migration was quantified using closure of scratch wound assays, contraction was assessed using attached and detached collagen lattices and extracellular matrix related gene expression using Taqman Realtime polymerase chain reaction. RESULTS: Human gingival fibroblasts isolated from both biotypes showed similar rates of closure of scratch wounds, which was not influenced by the addition of TGF-ß1 or TNFα. Fibroblasts from both biotypes contracted detached, but not attached, collagen gels to 50 % of their original weight although this contraction was not associated with incorporation of α-smooth muscle actin into stressfibres under any tested culture condition. Analysis of gene expression showed that POSTN, and ACTA2 mRNA levels did not significantly change, but CCN2 and COL1A2 mRNA levels were significantly higher in thick compared to thin fibroblasts in response to TGF-ß1. CONCLUSION: While supra-cellular factors influence the healing, esthetic outcomes and recession in thin gingival biotypes, differences in gingival fibroblast gene expression in response to growth factors may also play a role and warrants further investigation.

Lineage tracing of Foxd1-expressing embryonic progenitors to assess the role of divergent embryonic lineages on adult dermal fibroblast function.

Recent studies have highlighted the functional diversity of dermal fibroblast populations in health and disease, with part of this diversity linked to fibroblast lineage and embryonic origin. Fibroblasts derived from foxd1-expressing progenitors contribute to the myofibroblast populations present in lung and kidney fibrosis in mice but have not been investigated in the context of dermal wound repair. Using a Cre/Lox system to genetically track populations derived from foxd1-expressing progenitors, lineage-positive fibroblasts were identified as a subset of the dermal fibroblast population. During development, lineage-positive cells were most abundant within the dorsal embryonic tissues, contributing to the developing dermal fibroblast population, and remaining in this niche into adulthood. In adult mice, assessment of fibrosis-related gene expression in lineage-positive and lineage-negative populations isolated from wounded and unwounded dorsal skin was performed, identifying an enrichment of transcripts associated with matrix synthesis and remodeling in the lineage-positive populations. Using a novel excisional wound model, ventral skin healed with a greatly reduced frequency of foxd1 lineage-positive cells. This work supports that the embryonic origin of fibroblasts is an important predictor of fibroblast function, but also highlights that within disparate regions, fibroblasts of different lineages likely undergo convergent differentiation contributing to phenotypic similarities.

Low-flow intussusception and metastable VEGFR2 signaling launch angiogenesis in ischemic muscle.

Efforts to promote sprouting angiogenesis in skeletal muscles of individuals with peripheral artery disease have not been clinically successful. We discovered that, contrary to the prevailing view, angiogenesis following ischemic muscle injury in mice was not driven by endothelial sprouting. Instead, real-time imaging revealed the emergence of wide-caliber, primordial conduits with ultralow flow that rapidly transformed into a hierarchical neocirculation by transluminal bridging and intussusception. This process was accelerated by inhibiting vascular endothelial growth factor receptor-2 (VEGFR2). We probed this response by developing the first live-cell model of transluminal endothelial bridging using microfluidics. Endothelial cells subjected to ultralow shear stress could reposition inside the flowing lumen as pillars. Moreover, the low-flow lumen proved to be a privileged location for endothelial cells with reduced VEGFR2 signaling capacity, as VEGFR2 mechanosignals were boosted. These findings redefine regenerative angiogenesis in muscle as an intussusceptive process and uncover a basis for its launch.

Directional change produced by perpendicularly-oriented microgrooves is microtubule-dependent for fibroblasts and epithelium.

Anisotropic substrata such as micromachined grooves can control cell shape, orientation, and the direction of cell movement, a phenomena termed topographic guidance. Although many types of cells exhibit topographic guidance, little is known regarding cell responses to conflicting topographic cues. We employed a substratum with intersecting grooves in order to present fibroblasts and epithelial cells with conflicting topographic cues. Using time-lapse and confocal microscopy, we examined cell behavior at groove intersections. Migrating fibroblasts and epithelial cells typically extended a cell process into the intersection ahead of the cell body. After travelling along the "X" groove to enter the intersection, the leading lamellipodia of the cell body encountered the perpendicular "Y" groove, and spread latterly along the "Y" groove. The formation of lateral lamellipodia resulted in cells forming "T" or "L" morphologies, which were characterized by the formation of phosphotyrosine-rich focal adhesions at the leading edges. The "Y" groove did not prove an absolute barrier to cell migration, particularly for epithelial cells. Analysis of cytoskeletal distribution revealed that F-actin bundles did not adapt closely to the groove patterns, but typically did align to either the "X" or "Y" grooves. In contrast microtubules (MT) adapted closely to the walls. Inhibition of microtubule nucleation attenuated fibroblast and epithelial cell orientation within the intersection of the perpendicular grooves. We conclude that MT may be the prime determinant of fibroblast and epithelial cell conformation to conflicting topographies.

Periostin and matrix stiffness combine to regulate myofibroblast differentiation and fibronectin synthesis during palatal healing.

Although the matricellular protein periostin is prominently upregulated in skin and gingival healing, it plays contrasting roles in myofibroblast differentiation and matrix synthesis respectively. Palatal healing is associated with scarring that can alter or restrict maxilla growth, but the expression pattern and contribution of periostin in palatal healing is unknown. Using periostin-knockout (Postn-/-) and wild-type (WT) mice, the contribution of periostin to palatal healing was investigated through 1.5 mm full-thickness excisional wounds in the hard palate. In WT mice, periostin was upregulated 6 days post-wounding, with mRNA levels peaking at day 12. Genetic deletion of periostin significantly reduced wound closure rates compared to WT mice. Absence of periostin reduced mRNA levels of pivotal genes in wound repair, including α-SMA/acta2, fibronectin and ßigh3. Recruitment of fibroblasts and inflammatory cells, as visualized by immunofluorescent staining for fibroblast specific factor-1, vimentin, and macrophages markers Arginase-1 and iNOS was also impaired in Postn-/-, but not WT mice. Palatal fibroblasts isolated from the hard palate of mice were cultured on collagen gels and prefabricated silicon substrates with varying stiffness. Postn-/- fibroblasts showed a significantly reduced ability to contract a collagen gel, which was rescued by the exogenous addition of recombinant periostin. As the stiffness increased, Postn-/- fibroblasts increasingly differentiated into myofibroblasts, but not to the same degree as the WT. Pharmacological inhibition of Rac rescued the deficient myofibroblastic phenotype of Postn-/- cells. Low stiffness substrates (0.2 kPa) resulted in upregulation of fibronectin in WT cells, an effect which was significantly reduced in Postn-/- cells. Quantification of immunostaining for vinculin and integrinß1 adhesions revealed that Periostin is required for the formation of focal and fibrillar adhesions in mPFBs. Our results suggest that periostin modulates myofibroblast differentiation and contraction via integrinß1/RhoA pathway, and fibronectin synthesis in an ECM stiffness dependent manner in palatal healing.

Obstruction of Small Arterioles in Patients with Critical Limb Ischemia due to Partial Endothelial-to-Mesenchymal Transition.

Critical limb ischemia (CLI) is a hazardous manifestation of atherosclerosis and treatment failure is common. Abnormalities in the arterioles might underlie this failure but the cellular pathobiology of microvessels in CLI is poorly understood. We analyzed 349 intramuscular arterioles in lower limb specimens from individuals with and without CLI. Arteriolar densities were 1.8-fold higher in CLI muscles. However, 33% of small (<20 µm) arterioles were stenotic and 9% were completely occluded. The lumens were closed by bulky, re-oriented endothelial cells expressing abundant N-cadherin that uniquely localized between adjacent and opposing endothelial cells. S100A4 and SNAIL1 were also expressed, supporting an endothelial-to-mesenchymal transition. SMAD2/3 was activated in occlusive endothelial cells and TGFß1 was increased in the adjacent mural cells. These findings identify a microvascular closure process based on mesenchymal transitions in a hyper-TGFß environment that may, in part, explain the limited success of peripheral artery revascularization procedures.

Fibronectin synthesis, but not α-smooth muscle expression, is regulated by periostin in gingival healing through FAK/JNK signaling.

During skin healing, periostin facilitates myofibroblast differentiation through a ß1 integrin/FAK dependent mechanism and continued expression is associated with scarring. In contrast to skin, gingival tissue does not typically scar upon injury, but the role of periostin in gingival healing has never been investigated. Using a rat gingivectomy model, we show that the gingival architecture is re-established within 14 days of wounding. Periostin mRNA levels peak at day 7 post-wounding, with persistence of periostin protein in the connective tissue through day 14. Collagen type I and lysyl oxidase mRNA levels peak at day 7 post wounding, which corresponded with the peak of fibroblast proliferation. Although α-smooth muscle actin mRNA levels increased 200-fold in the tissue, no myofibroblasts were detected in the regenerating tissue. In vitro, human gingival fibroblast adhesion on periostin, but not collagen, was inhibited by blocking ß1 integrins. Fibroblasts cultured on periostin exhibited similar rates of proliferation and myofibroblast differentiation to cells cultured on collagen only. However, human gingival fibroblasts cultured in the presence of periostin exhibited significantly increased fibronectin and collagen mRNA levels. Increases in fibronectin production were attenuated by pharmacological inhibition of FAK and JNK signaling in human gingival fibroblasts. In vivo, mRNA levels for fibronectin peaked at day 3 and 7 post wounding, with protein immunoreactivity highest at day 7, suggesting periostin is a modulator of fibronectin production during gingival healing.

Matrix composition in 3-D collagenous bioscaffolds modulates the survival and angiogenic phenotype of human chronic wound dermal fibroblasts.

There is a substantial need for new strategies to stimulate cutaneous tissue repair in the treatment of chronic wounds. To address this challenge, our team is developing modular biomaterials termed "bead foams", comprised of porous beads synthesized exclusively of extracellular matrix (ECM) and assembled into a cohesive three-dimensional (3-D) network. In the current study, bead foams were fabricated from human decellularized adipose tissue (DAT) or commercially-sourced bovine tendon collagen (COL) to explore the effects of ECM composition on human wound edge dermal fibroblasts (weDF) sourced from chronic wound tissues. The DAT and COL bead foams were shown to be structurally similar, but compositionally distinct, containing different levels of glycosaminoglycan content and collagen types IV, V, and VI. In vitro testing under conditions simulating stresses within the chronic wound microenvironment indicated that weDF survival and angiogenic marker expression were significantly enhanced in the DAT bead foams as compared to the COL bead foams. These findings were corroborated through in vivo assessment in a subcutaneous athymic mouse model. Taken together, the results demonstrate that weDF survival and paracrine function can be modulated by the matrix source applied in the design of ECM-derived scaffolds and that the DAT bead foams hold promise as cell-instructive biological wound dressings. STATEMENT OF SIGNIFICANCE: Biological wound dressings derived from the extracellular matrix (ECM) can be designed to promote the establishment of a more permissive microenvironment for healing in the treatment of chronic wounds. In the current work, we developed modular biomaterials comprised of fused networks of porous ECM-derived beads fabricated from human decellularized adipose tissue (DAT) or commercially-available bovine collagen. The bioscaffolds were designed to be structurally similar to provide a platform for investigating the effects of ECM composition on human dermal fibroblasts isolated from chronic wounds. Testing in in vitro and in vivo models demonstrated that cell survival and pro-angiogenic function were enhanced in the adipose-derived bioscaffolds, which contained higher levels of glycosaminoglycans and collagen types IV, V, and VI. Our findings support that the complex matrix composition within DAT can induce a more pro-regenerative cellular response for applications in wound healing.

Periostin and CCN2 Scaffolds Promote the Wound Healing Response in the Skin of Diabetic Mice.

IMPACT STATEMENT: Nonhealing skin wounds remain a significant burden on health care systems, with diabetic patients 20 times as likely to undergo a lower extremity amputation due to impaired healing. Novel treatments that suppress the proinflammatory signature and induce the proliferative and remodeling phases are needed clinically. We demonstrate that the addition of periostin and CCN2 in a scaffold form increases closure rates of full-thickness skin wounds in diabetic mice, concomitant with enhanced angiogenesis. Our results demonstrate the efficacy of periostin- and CCN2-containing biomaterials to stimulate wound closure, which could represent a novel method for the treatment of diabetic skin wounds.