Fetal wound healing: a biochemical study of scarless healing

Human fetal surgery is being successfully performed today in a small number of highly selected patients for conditions that may lead to irreversible damage to the fetus and threaten the viability of the newborn. Following surgical repair, fetal wounds heal without scarring. This study was initiated to characterize fetal wounds both histologically and biochemically. Gore-Tex tubing was implanted into the subcutaneous tissue of the back of fetal, newborn, and adult New Zealand white rabbits. Light microscopic examination of healed wounds revealed no evidence of scar formation. Electron microscopy demonstrated a striated fibrillar structure suggestive of collagen within the lumen of the Gore-Tex tubing implants. Amino acid analysis (sensitivity 40 pmol) confirmed the presence of hydroxylysine and hydroxyproline within the Gore-Tex wound chambers indicating the presence of collagen in fetal wounds. The small amount of collagen precluded the typing of the collagen using cyanogen bromide peptide analysis. The absence of scarring and the small amounts of detectable collagen suggest a high degree of reorganization of the connective tissues involved in repair. The fetal wound matrix is rich in hyaluronic acid. Topical hyaluronic acid has been associated experimentally with a reduced amount of scarring in postnatal wound healing. Hyaluronic acid extracted from human skin and scar tissue is associated with collagen and other proteins. We propose that a hyaluronic acid-collagen-protein complex may play a role in fetal wound healing.     

Scarless skin wound repair in the fetus.

The ability of a fetus to heal without scar formation depends on its gestational age at the time of injury and the size of the wound defect. In general, linear incisions heal without scar until late in gestation whereas excisional wounds heal with scar at an earlier gestational age. The profiles of fetal proteoglycans, collagens, and growth factors are different from those in adult wounds. The less-differentiated state of fetal skin is probably an important characteristic responsible for scarless repair. There is minimal inflammation in fetal wounds. Fetal wounds are characterized by high levels of hyaluronic acid and its stimulator(s) with more rapid, highly organized collagen deposition. The roles of peptide growth factors such as transforming growth factor-beta and basic fibroblast growth factor are less prominent in fetal than in adult wound healing. Platelet-derived growth factor has been detected in scarless fetal skin wounds, but its role is unknown. An understanding of scarless tissue repair has possible clinical application in the modulation of adult fibrotic diseases and abnormal scar-forming conditions.     

Lentiviral-mediated over-expression of hyaluronan synthase-1 (HAS-1) decreases the cellular inflammatory response and results in regenerative wound repair

Fetal wounds have been found to have increased levels of high-molecular-weight hyaluronan (HMW-HA) compared with those of adults. The primary enzyme responsible for producing HMW-HA is hyaluronic acid synthase-1 (HAS-1). We hypothesized that over-expression of HAS-1 in adult dermal wounds would decrease inflammation and promote regenerative healing. To test this hypothesis, the flanks of adult C57Bl/6 mice were treated with a lentiviral construct containing either HAS-1-GFP or GFP transgenes. After 48 h, a 4-mm excisional wound was made at the site of treatment. Wounds were harvested at days 3, 7, or 28 after wounding. Wound phenotype was assessed by histology to examine tissue architecture and immunohistochemistry for CD45. At 7 and 28 days, lenti-HAS-1-treated wounds demonstrated the restoration of the normal dermal elements and organized collagen fiber orientation. In contrast, the lenti-GFP-treated wounds lacked normal dermal architecture and demonstrated a disorganized collagen scar. At 3 and 7 days, wounds treated with lenti-HAS-1 exhibited a significant decrease in the number of inflammatory cells when compared with wounds treated with lenti-GFP. Thus, HAS-1 over-expression promotes dermal regeneration, in part by decreasing the inflammatory response and by recapitulation of fetal extracellular matrix HMW-HA content.     

A comparative analysis of healing of surgical cleft lip corrected in utero and neonates.

We studied the healing process in surgically created cleft lips in fetal mice and compared it with that in newborn mice with cleft lips. Our purpose was to determine the time for optimal healing, defined as minimal scarring, for a repaired cleft lip. Full-thickness paramedian lip incisions were made in NMRI mice in utero, in 2- and 4-day-old neonates, and in adults (n = 10 in each experimental and control group). The healing process was studied by biochemical analysis of hyaluronic acid and hydroxyproline content in the repaired cleft tissue. We found that the production of hyaluronic acid remained stable during the healing period and was similar in all experimental groups. However, there was an unexplained but consistent depression in the hyaluronic acid content of fetal tissue 2 days after repair. Hydroxyproline was present in the fetal healing tissue, but in a low concentration, starting 4 days after surgical incision of the lip. The production of hydroxyproline in 2-day-old neonates was similar to that in the fetuses throughout the healing period (p less than 0.0005). However, the production of hydroxyproline increased in 4-day-old neonatal and adult tissues. In conclusion, we found an optimal healing period for mice with minimal collagen production in the late fetal stage, and this lasted 2 days after birth.     

Regeneration of corneal tissue

Penetrating wounds in rabbit corneas heal to form an opaque tissue that eventually becomes transparent. DNA content, dry weight, water content, and collagen content of the tissue gradually become more like that of normal cornea. The healing tissues also synthesize low-sulfated keratan sulfate, hyaluronic acid, and heparan sulfate. These glycosaminoglycans are not found in normal adult corneas but have been reported in fetal corneas. Previous studies have shown that collagen from healing corneal wounds and collagen from fetal corneas have very similar cross-linking patterns, but these patterns are different from those in normal adult collagen. The similarities between collagen and glycosaminoglycans in healing corneal wounds and in fetuses suggest some recapitulation of ontogenetic processes. The biochemical sequence and eventual return of transparency to the rabbit cornea indicate a capability for true regeneration of stromal tissue in the rabbit.     

Nerve dependency in scarless fetal wound healing

The human fetus is capable of healing cutaneous wounds without scar up to the third trimester of development This process of tissue repair is more akin to newt limb regeneration than classic adult scar forming wound repair. Regeneration of the newt limb is dependent on neural input in its early stages. This study was an attempt to determine whether a similar dependence on neural input exists for mammalian fetal wounds to heal without scar. The left hind limb of six fetal lambs was denervated during the early second trimester of development (day 55; term = 145 days). Two weeks after denervation, the animals were again exposed to create bilateral incisional and 6-mm-diameter excisional wounds on their innervated right and denervated left lower extremities. Five days after creation of these defects, the wounds were examined for alterations in repair. Four fetal lambs survived, and three were suitable for evaluation. There were marked alterations in wound healing seen after denervation. Excisional wounds on the innervated side contracted and decreased their surface area by 14 percent. In contrast, the denervated wounds not only failed to contract, but increased in size by 60 percent. Changes in the incisional wounds were equally distinctive. Innervated incisional wounds healed completely without scar and had a wound breaking strength comparable to that of normal skin (Table I). In contrast, two of the three denervated incisional wounds dehisced and failed to heal, even in the regions where the skin was approximated by suture. The third denervated incisional wound did heal but with a significant amount of scar. Electron microscopy confirmed this finding by clearly demonstrating thickened and irregular collagen deposition in the extracellular matrix of all the denervated incisional specimens. In summary, like the regenerating newt limb, scarless fetal skin wound repair requires neural stimulation for tissue regeneration to occur. Therefore, in the mammal, the primary regulator for this unique type of tissue repair may have a central neural, rather than a local, tissue origin.     

Immunohistochemical localization of growth factors in fetal wound healing

Fetal wound healing occurs rapidly, in a regenerative fashion, and without scar formation, by contrast with adult wound healing, where tissue repair results in scar formation which limits tissue function and growth. The extracellular matrix deposited in fetal wounds contains essentially the same structural components as that in the adult wound but there are distinct differences in the spatial and temporal distribution of these components. In particular the organization of collagen in the healed fetal wound is indistinguishable from the normal surrounding tissue. Rapidity of healing, lack of an inflammatory response, and an absence of neovascularization also distinguish fetal from adult wound healing. The mechanisms controlling these differing processes are undefined but growth factors may play a critical role. The distribution of growth factors in healing fetal wounds is unknown. We have studied, by immunohistochemistry, the localization of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta), and basic fibroblast growth factor (bFGF), in fetal, neonatal, and adult mouse lip wounds. TGF beta and bFGF were present in neonatal and adult wounds, but were not detected in the fetal wounds, while PDGF was present in fetal, neonatal, and adult wounds. This pattern correlates with the known effects in vitro of these factors, the absence of an inflammatory response and neovascularization in the fetal wound, and the patterns of collagen deposition in both fetal and adult wounds. The results suggest that it may be possible to manipulate the adult wound to produce more fetal-like, scarless, wound healing.     

Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition.

Fetal wounds pass from scarless repair to healing with scar formation during gestation. This transition depends on both the size of the wound and the gestational age of the fetus. This study defines the transition period in the fetal rat model and provides new insight into scarless collagen wound architecture by using confocal microscopy. A total of 16 pregnant Sprague-Dawley rats were operated on. Open full-thickness wounds, 2 mm in diameter, were created on fetal rats at gestational ages 14.5 days (E14; n = 10), 16.5 days (E16; n = 42), and 18.5 days (E18; n = 42) (term = 21.5 days). Wounds were harvested at 24 (n = 18 per gestational age) and 72 hours (n = 24 per gestational age). Skin at identical gestational ages to wound harvest was used for controls. The wounds were fixed and stained with hematoxylin and eosin, antibody to type I collagen, and Sirius red for confocal microscopic evaluation. No E14 rat fetuses survived to wound harvest. Wounds created on E16 fetal rats healed completely and without scarring. E16 fetal rat hair follicle formation and collagen architecture was similar to that of normal, nonwounded skin. Wounds created on E18 fetal rats demonstrated slower healing; only 50 percent were completely healed at 72 hours compared with 100 percent of the E16 fetal rat wounds at 72 hours. Furthermore, the E18 wounds healed with collagen scar formation and without hair follicle formation. Confocal microscopy demonstrated that the collagen fibers were thin and arranged in a wispy pattern in E16 fetal rat wounds and in nonwounded dermis. E18 fetal rat wounds had thickened collagen fibers with large interfiber distances. Two-millimeter excisional E16 fetal rat wounds heal without scar formation and with regeneration of normal dermal and epidermal appendage architecture. E18 fetal rat wounds heal in a pattern similar to that of adult cutaneous wounds, with scar formation and absence of epidermal appendages. Confocal microscopy more clearly defined the dermal architecture in normal skin, scarless wounds, and scars. These data further define the transition period in the fetal rat wound model, which promises to be an effective system for the study of in vivo scarless wound healing.     

Collagen-binding peptidoglycans inhibit MMP mediated collagen degradation and reduce dermal scarring

Scarring of the skin is a large unmet clinical problem that is of high patient concern and impact. Wound healing is complex and involves numerous pathways that are highly orchestrated, leaving the skin sealed, but with abnormal organization and composition of tissue components, namely collagen and proteoglycans, that are then remodeled over time. To improve healing and reduce or eliminate scarring, more rapid restoration of healthy tissue composition and organization offers a unique approach for development of new therapeutics. A synthetic collagen-binding peptidoglycan has been developed that inhibits matrix metalloproteinase-1 and 13 (MMP-1 and MMP-13) mediated collagen degradation. We investigated the synthetic peptidoglycan in a rat incisional model in which a single dose was delivered in a hyaluronic acid (HA) vehicle at the time of surgery prior to wound closure. The peptidoglycan treatment resulted in a significant reduction in scar tissue at 21 days as measured by histology and visual analysis. Improved collagen architecture of the treated wounds was demonstrated by increased tensile strength and transmission electron microscopy (TEM) analysis of collagen fibril diameters compared to untreated and HA controls. The peptidoglycan's mechanism of action includes masking existing collagen and inhibiting MMP-mediated collagen degradation while modulating collagen organization. The peptidoglycan can be synthesized at low cost with unique design control, and together with demonstrated preclinical efficacy in reducing scarring, warrants further investigation for dermal wound healing.     

The extracellular matrix of lip wounds in fetal, neonatal and adult mice.

Wound healing in the fetus occurs rapidly, by a regenerative process and without an inflammatory response, resulting in complete restitution of normal tissue function. By contrast, in the adult, wounds heal with scar formation, which may impair function and inhibit further growth. The cellular mechanisms underlying these differing forms of wound healing are unknown but the extracellular matrix (ECM), through its effects on cell function, may play a key role. We have studied the ECM in upper lip wounds of adult, neonatal and fetal mice at days 14, 16 and 18 of gestation. The spatial and temporal distribution of collagen types I, III, IV, V and VI, fibronectin, tenascin, laminin, chondroitin and heparan sulphates were examined immunohistochemically. Results from the fetal groups were essentially similar whilst there were distinct differences between fetus, neonate and adult. Fibronectin was present at the surface of the wound in all groups at 1 h post-wounding. Tenascin was also present at the wound surface but the time at which it was first present differed between fetus (1 h), neonate (12 h) and adult (24 h). The time of first appearance paralleled the rate of wound healing which was most rapid in the fetus and slowest in the adult. Tenascin inhibits the cell adhesion effect of fibronectin and during development the appearance of tenascin correlates with the initiation of cell migration. During wound healing the appearance of tenascin preceded cell migration and the rapid closure of fetal wounds may be due to the early appearance of tenascin in the wound. Collagen types I, III, IV, V and VI were present in all three wound groups but the timing and pattern of collagen deposition differed, with restoration of the normal collagen pattern in the fetus and a scar pattern in the adult. This confirms that lack of scarring in fetal wounds is due to the organisation of collagen within the wound and not simply lack of collagen formation. The distribution of chondroitin sulphate differed between normal fetal and adult tissues and between fetal and adult wounds. Its presence in the fetal wound may alter collagen fibril formation. No inflammatory response was seen in the fetal wounds. The differences in the ECM of fetal and adult wounds suggests that it may be possible to alter the adult wound so that it heals by a fetal-like process without scar formation, loss of tissue function or restriction of growth.     

Skin Wound Healing and Scarring: Fetal Wounds and Regenerative Restitution

The adverse physiological and psychological effects of scars formation after healing of wounds are broad and a major medical problem for patients. In utero, fetal wounds heal in a regenerative manner, though the mechanisms are unknown. Differences in fetal scarless regeneration and adult repair can provide key insight into reduction of scarring therapy. Understanding the cellular and extracellular matrix alterations in excessive adult scarring in comparison to fetal scarless healing may have important implications. Herein, we propose that matrix can be controlled via cellular therapy to resemble a fetal‐like matrix that will result in reduced scarring. Birth Defects Research (Part C) 96:325–333, 2012. © 2013 Wiley Periodicals, Inc.     

Quantitative characteristics of changes in the cellular composition and vascularization of aseptic wounds in rat skin, healing without treatment and during stimulation of repair processes by exogenous collagen

The changes in cellular composition and vascularization of aseptic wounds on the rat skin were assessed quantitatively using the ocular net without treatment and during stimulation of repair processes by exogenous collagen. An intensive increase in the number of macrophages, endotheliocytes and fibroblasts was observed in wounds without treatment by the fifth day, with maximum vascularization of the granulation tissue occurring by the seventh day. During stimulation of repair processes by collagen the macrophage reaction, proliferation of endotheliocytes and fibroblasts and vascularization of wounds were activated earlier, while the stereotype relationships of the cellular components remained unchanged. The intercellular relationships of the wound healing process are discussed.     

CD44-dependent inflammation,fibrogenesis, and collagenolysis regulates extracellular matrix remodeling and tensile strength during cutaneous wound healing

Cutaneous wound healing consists of three main phases: inflammation, re-epithelialization, and tissue remodeling. During normal wound healing, these processes are tightly regulated to allow restoration of skin function and biomechanics. In many instances, healing leads to an excess accumulation of fibrillar collagen (the principal protein found in the extracellular matrix - ECM), and the formation of scar tissue, which has compromised biomechanics, tested using ramp to failure tests, compared to normal skin (Corr and Hart, 2013 [1]). Alterations in collagen accumulation and architecture have been attributed to the reduced tensile strength found in scar tissue (Brenda et al., 1999; Eleswarapu et al., 2011). Defining mechanisms that govern cellular functionality and ECM remodeling are vital to understanding normal versus pathological healing and developing approaches to prevent scarring. CD44 is a cell surface adhesion receptor expressed on nearly all cell types present in dermis. Although CD44 has been implicated in an array of inflammatory and fibrotic processes such as leukocyte recruitment, T-cell extravasation, and hyaluronic acid (the principal glycosaminoglycan found in the ECM) metabolism, the role of CD44 in cutaneous wound healing and scarring remains unknown. We demonstrate that in an excisional biopsy punch wound healing model, CD44-null mice have increased inflammatory and reduced fibrogenic responses during early phases of wound healing. At wound closure, CD44-null mice exhibit reduced collagen degradation leading to increased accumulation of fibrillar collagen, which persists after wound closure leading to reduced tensile strength resulting in a more severe scarring phenotype compared to WT mice. These data indicate that CD44 plays a previously unknown role in fibrillar collagen accumulation and wound healing during the injury response.     

Fibronectin involvement in granulation tissue and wound healing in rabbits

This study describes the distribution of fibronectin and its association with reticulin fibers (type III collagen) and hyaluronic acid in shallow rabbit wounds. Linear incisions were made dorsally with a surgical blade. Animals were sacrificed and 1,2,3,4,5, and 8 day wounds were examined using peroxidase-antiperoxidase to localize affinity-purified antibodies to fibronectin. Tissue samples were also stained with hematoxylin and eosin in addition to silver stains for reticulin, and Alcian blue for hyaluronic acid. After wounding, the incision filled with a fibrin clot that stained positively for fibronectin. The underlying dermis and adjacent, unwounded dermis also contained fibronectin. Epidermal cells that migrate from the wound margin between the clot and the dermis were in direct association with fibronectin in these wound components. By 72 hr, epidermal continuity was reestablished. Early granulation tissue formation was apparent just below the epidermis 5 day wounds. Fibronectin was observed in the matrix surrounding individual fibroblasts and codistributed with reticulin fibers and hyaluronic acid in both 5 and 8 day wounds. Granulation tissue of 8 day wounds stained intensely for fibronectin and extended to a greater depth in the reticular dermis. Dense fibrillar networks of fibronectin and fibroblasts were aligned parallel to the epidermis, giving the granulation tissue a highly structured and organized appearance. Fibroblasts contained fibronectin and were surrounded by less fibronectin at the wound periphery than within the granulation tissue. These findings suggest that fibronectin may be important in the reconstruction of tissues during repair by functioning as an extracellular scaffold for migrating cells.     

Scarless fetal wound healing: a basic science review

SUMMARY: Scar formation is a major medical problem that can have devastating consequences for patients. The adverse physiological and psychological effects of scars are broad, and there are currently no reliable treatments to prevent scarring. In contrast to adult wounds, early gestation fetal skin wounds repair rapidly and in the absence of scar formation. Despite extensive investigation, the exact mechanisms of scarless fetal wound healing remain largely unknown. For some time, it has been known that significant differences exist among the extracellular matrix, inflammatory response, cellular mediators, and gene expression profiles of fetal and postnatal wounds. These differences may have important implications in scarless wound repair.     

Matrix metalloproteinases and the ontogeny of scarless repair: the other side of the wound healing balance

Early gestation mammalian fetuses possess the remarkable ability to heal cutaneous wounds in a scarless fashion. Over the past 20 years, scientists have been working to decipher the mechanisms underlying this phenomenon. Much of the research to date has focused on fetal correlates of adult wound healing that promote fibrosis and granulation tissue formation. It is important to remember, however, that wound repair consists of a balance between tissue synthesis, deposition, and degradation. Relatively little attention has been paid to this latter component of the fetal wound healing process.In this study, we examined the ontogeny of ten matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in nonwounded fetal rat skin and fibroblasts as a function of gestational age. We used a semiquantitative polymerase chain reaction protocol to analyze these important enzymes at time points that represent both the scarless and scar-forming periods of rat gestation. The enzymes evaluated were collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3, and TIMPs 1, 2, and 3.Results demonstrated marked increases in gene expression for MMP-1, MMP-3 and MMP-9 that correlated with the onset of scar formation in nonwounded fetal skin. Similar results were noted in terms of MMP-9 gene expression in fetal fibroblasts. These results suggest that differences in the expression of these matrix metalloproteinases may have a role in the scarless wound healing phenotype observed early in fetal rat gestation. Furthermore, our data suggest that the differential expression of gelatinase B (MMP-9) may be mediated by the fetal fibroblasts themselves.     

Role of platelet-derived growth factor in wound healing

Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. PDGF stimulates chemotaxis, proliferation, and new gene expression in monocytes-macrophages and fibroblasts in vitro, cell types considered essential for tissue repair. Therefore, we analyzed the influence of exogenously administered recombinant B chain homodimers of PDGF (PDGF-BB) on two experimental tissue repair paradigms, incisional and excisional wounds. In both types of wounds, as little as 20-200 picomoles applied a single time to wounds significantly augmented the time dependent influx of inflammatory cells and fibroblasts and accelerated provisional extracellular matrix deposition and subsequent collagen formation. In incisional wounds, PDGF-BB augmented wound breaking strength 50-70% over the first 3 weeks; in excisional wounds, PDGF-BB accelerated time to closure by 30%. PDGF-BB exaggerated, but did not alter, the normal course of soft tissue repair, resulting in a significant acceleration of healing. Long term observations established no apparent differences between PDGF-BB treated and non-treated wounds. Thus, the vulnerary effects of PDGF-BB were transient and fully reversible in both wound healing models. Furthermore, analysis of PDGF-treated and non-treated wounds has provided important insights into mechanisms of normal and deficient tissue repair processes. PDGF appears to transduce its signal through wound macrophages and may trigger the induction of positive autocrine feedback loops and synthesis of endogenous wound PDGF and other growth factors, thereby enhancing the cascade of tissue repair processes required for a fully-healed wound. Thus, PDGF and other wound produced polypeptide growth factors may be the critical regulators of extracellular matrix deposition within healing wounds.     

The role of oxidised regenerated cellulose/collagen in chronic wound repair and its potential mechanism of action

Normal wound healing is a carefully controlled balance of destructive processes necessary to remove damaged tissue and repair processes which lead to new tissue formation. Proteases and growth factors play a pivotal role in regulating this balance, and if disrupted in favour of degradation then delayed healing ensues; a trait of chronic wounds. Whilst there are many types of chronic wounds, biochemically they are thought to be similar in that they are characterised by a prolonged inflammatory phase, which results in elevated levels of proteases and diminished growth factor activity. This increase in proteolytic activity and subsequent degradation of growth factors is thought to contribute to the net tissue loss associated with these chronic wounds.

In this study, we describe a new wound treatment, comprising oxidised regenerated cellulose and collagen (ORC/collagen), which can redress this imbalance and modify the chronic wound environment. We demonstrate that ORC/collagen can inactivate potentially harmful factors such as proteases, oxygen free radicals and excess metal ions present in chronic wound fluid, whilst simultaneously protecting positive factors such as growth factors and delivering them back to the wound.

These characteristics suggest a beneficial role for this material in helping to re-balance the chronic wound environment and therefore promote healing.     

Adherence,proliferation and collagen turnover by human fibroblasts seeded into different types of collagen sponges

We describe an in vitro model that we have used to evaluate dermal substitutes and to obtain data on cell proliferation, the rate of degradation of the dermal equivalent, contractibility and de novo synthesis of collagen. We tested three classes of collagenous materials: (1) reconstituted non-crosslinked collagen, (2) reconstituted collagen that was chemically crosslinked with either glutaraldehyde, aluminium alginate or acetate, and (3) native collagen fibres, with or without other extracellular matrix molecules (elastin hydrolysate, hyaluronic acid or fibronectin). The non-crosslinked reconstituted collagen was degraded rapidly by human fibroblasts. Teh chemically crosslinked materials proved to be cytotoxic. Native collagen fibres were stable. In the absence of ascorbic acid, the addition of elastin hydrolysate to this type of matrix reduced the rate of collagen degradation. Both elastin hydrolysate and fibronectin partially prevented fibroblast-mediated contraction. Hyaluronic acid was only slightly effective in reducing the collagen degradation rate and more fibroblast-mediated contraction of the material was found than for the native collagen fibres with elastin hydrolysate and fibronectin. In the presence of ascorbate, collagen synthesis was enhanced in the native collagen matrix without additions and in the material containing elastin hydrolysate, but not in the material with hyaluronic acid. These results are indicative of the suitability of tissue substitutes for in vivo application.     

Role of collagen XII in skin homeostasis and repair

Skin integrity and function depends to a large extent on the composition of the extracellular matrix, which regulates tissue organization. Collagen XII is a homotrimer with short collagenous domains that confer binding to the surface of collagen I-containing fibrils and extended flexible arms, which bind to non-collagenous matrix components. Thereby, collagen XII helps to maintain collagen suprastructure and to absorb stress. Mutant or absent collagen XII leads to reduced muscle and bone strength and lax skin, whereas increased collagen XII amounts are observed in tumor stroma, scarring and fibrosis.This study aimed at uncovering in vivo mechanisms by which collagen XII may achieve these contrasting outcomes. We analyzed skin as a model tissue that contains abundant fibrils, composed of collagen I, III and V with collagen XII decorating their surface, and which is subject to mechanical stress. The impact of different collagen XII levels was investigated in collagen XII-deficient (Col12-KO) mice and in mice with collagen XII overexpression in the dermis (Col12-OE). Unchallenged skin of these mice was histologically inconspicuous, but at the ultrastructural level revealed distinct aberrations in collagen network suprastructure. Repair of excisional wounds deviated from controls in both models by delayed healing kinetics, which was, however, caused by completely different mechanisms in the two mouse lines. The disorganized matrix in Col12-KO wounds failed to properly sequester TGFβ, resulting in elevated numbers of myofibroblasts. These are, however, unable to contract and remodel the collagen XII-deficient matrix. Excess of collagen XII, in contrast, promotes persistence of M1-like macrophages in the wound bed, thereby stalling the wounds in an early inflammatory stage of the repair process and delaying healing.Taken together, we demonstrate that collagen XII is a key component that assists in orchestrating proper skin matrix structure, controls growth factor availability and regulates cellular composition and function. Together, these functions are pivotal for re-establishing homeostasis after injury.