Many cells and soluble factors are involved in the wound healing process, which can be divided into inflammatory, proliferative, and remodeling phases. Fibroblasts play a crucial role in wound healing, especially during the proliferative phase, and show heterogeneity depending on lineage, tissue distribution, and extent of differentiation. Fibroblasts from tissue stem cells rather than from healthy tissues infiltrate wounds and proliferate. Some fibroblasts in the wound healing site express the mesenchymal stem cell marker, p75NTR. In the cell culture system, fibroblasts attached to collagen fibrils stop growing, even in the presence of protein growth factors, thus mimicking the quiescent nature of fibroblasts in healthy tissues. Fibroblasts in wound healing sites proliferate and are surrounded by collagen fibrils. These facts indicate presence of new growth-initiating factor for fibroblasts attached to collagen fibrils at the wound healing site, where the collagen-derived peptide, prolyl-hydroxyproline (Pro-Hyp), is generated. Pro-Hyp triggers the growth of p75NTR-positive fibroblasts cultured on collagen gel but not p75NTR-negative fibroblasts. Thus, Pro-Hyp is a low molecular weight growth-initiating factor for specific fibroblasts that is involved in the wound healing process. Pro-Hyp is also supplied to tissues by oral administration of gelatin or collagen hydrolysate. Thus, supplementation of gelatin or collagen hydrolysate has therapeutic potential for chronic wounds. Animal studies and human clinical trials have demonstrated that the ingestion of gelatin or collagen hydrolysate enhances the healing of pressure ulcers in animals and humans and improves delayed wound healing in diabetic animals. Therefore, the low molecular weight fibroblast growth-initiating factor, Pro-Hyp, plays a significant role in wound healing and has therapeutic potential for chronic wounds.
Cutaneous wound healing is a complex and highly regulated process with many cells and soluble factors working together during this process. The cutaneous wound healing process is generally divided into three overlapped phases: inflammatory, proliferative, and remodeling or maturation phases. The inflammatory phase starts shortly after hemostasis and is triggered by the activation of adhered platelets. Chemokines and cytokines, which are released from the activated platelets and damaged tissue, recruit neutrophils and monocytes to the wound sites. Neutrophils, which appear in the wound 4 h after injury and decrease during the subsequent weeks, eliminate pathogens by phagocytosis and the release of bactericidal reactive oxygen species and peptides. The infiltration of monocytes starts the second day and the monocytes are changed to macrophages. Macrophages increase and reach maximum levels during the proliferative process. Macrophages scavenge tissue debris and the remaining neutrophils by phagocytosis and secrete protein growth factors and cytokines that promote proliferation and migration of the cells involved in wound healing. Circulating lymphocytes migrate to the wound healing sites early after injury and remain up to the last phase. Approximately 3 days after the initial injury, the proliferative phase starts. This phase is responsible for the lesion closure. Fibroblasts infiltrate to the wound sites and proliferate and produce extracellular compounds such as collagen, which form the basis of granulation tissue. The granulation tissue provides the basis for re-epithelization and some fibroblasts differentiate into myofibroblasts, initiating wound contraction. To supply nutrients to the granulation tissue, the induction of angiogenesis occurs. After ~2–3 weeks, the proliferative phase is followed by the remodeling or maturation phase to achieve tensile strength through contraction, reorganization, degradation, and resynthesis of the extracellular matrix. The cells involved in the previous phases are removed by apoptosis. Thus, the granulation tissue is gradually remodeled and changed to scar tissue consisting of less cellular and vascular components and rich in collagen fibers, resulting in an ~70–80% tensile strength of the uninjured skin. Aberrant wound healing may result in a chronic wound or non-healing wound, which is a burden to the patient, caregiver, and medical system. Aberrant wound healing is often occurs under diabetic and malnutrition conditions.
In wound healing process, fibroblasts play a significant role especially during the proliferative phase. Fibroblasts migrate, proliferate, and produce extracellular matrix compounds in response to protein growth factors, such as epidermal growth factor, insulin, interleukin-1β, tumor necrosis factor-α, transforming growth factor-β1, platelet-derived growth factor, and fibroblast growth factors. In cell culture systems, fibroblasts proliferate on plastic substrate in the presence of fetal bovine serum (FBS), which is rich in protein growth factors and fibronectin. The removal of low molecular weight compounds from FBS does not affect the growth of fibroblasts on the plastic substrate. Fibroblasts have to attach to the substrates for survival. In tissues, fibroblasts attach to collagen fibrils directly using collagen receptors such as α1β1 and α2b1 integrins and indirectly via fibronectin using α5β1 integrin. In cell culture systems using non-coated culture plates, fibroblasts are attached to the plate surface via fibronectin, which is present in FBS and promote cell proliferation. However, some researchers have used collagen gel-coated plates for the cultivation of fibroblasts. The collagen solution becomes to be polymerized at neutral pH after incubation at 37°C. During this process, collagen molecules assemble into collagen fibrils. Fibroblasts that are attached to the collagen fibrils get in a quiescent state even in the presence of FBS. The denatured collagen also forms gel but does not assemble into the fibrils, and cannot inhibit the growth of fibroblasts. A higher concentration of collagen gels induce a robust inhibitory effect on fibroblast proliferation. Therefore, the inhibitory effect of collagen gel on fibroblast growth cannot be solely attributed to softness of collagen gels compared to plastic plate, while it is known that extracellular mechanical stress affect the growth and differentiation of cells, including fibroblasts. Thus, fibroblasts growth is controlled not only by protein growth factors but also by interaction with collagen fibrils. Growth regulatory mechanisms of fibroblast settled on collagen fibrils are thus vital for understanding the in vivo wound healing process. Nevertheless, this topic has received limited attention many years up to now.
Normal fibroblasts present in a quiescent state all over the connective tissues in the body under physiological condition. The suppression of proliferation of fibroblast cultured on collagen gel could therefore mimic the quiescent characteristics of fibroblasts in healthy tissue. However, fibroblasts in collagen matrixes start to proliferate after wounding, in which, additional factor(s) are required for initiating fibroblast proliferation. However, rational information is still limited for the fibroblasts under such context. Our group have previously addressed; after total skin excision in mice, collagen di-peptide, prolyl-hydroxyproline (Pro-Hyp), which is generated in the granulation tissue after wounding and Pro-Hyp can act as a trigger for growth initiation in the fibroblasts cultured on collagen gel.
The present study does not aim to review cutaneous wound healing processes comprehensively. Instead, the present mini review is thus focusing especially on the function of a new low molecular weight growth-initiating factor, Pro-Hyp, in wound healing process and discuss its therapeutic potential for chronic wounds in the end.
Collagen is a major extracellular matrix protein and has a triple helical domain and small globular domains. Collagen forms a molecular family consisting of different gene products. Collagen molecular species are referred to as “Type” with Roman numerals. Types I, II, III, V, and XI collagens form collagen fibrils and are referred to as fibril-forming collagens. Other collagen types are classified as non-fibrillar collagen, which play essential biological functions; however, their contents are far lower than that of the fibrillar collagens. Type I collagen is the primary collagen in the skin, bone, and tendons. Type III and V collagens co-exist with Type I collagen. Type II collagen is the primary collagen in cartilage with minor Type XI collagen. Collagen molecules consist of three subunits referred to as the α chain. Each α chain is designated by Roman numerals (Type) and Arabic numerals (subunit number). The major collagen molecules in the skin, Types I and III collagens, are designated as [α1(I)]2 α2(I) and [α1(III)]3, respectively.
Collagen consists of post-translationally modified amino acids, hydroxyproline (Hyp), and hydroxylysine (Hyl), which play significant roles in stabilizing the triple helix structure and the inter- and intra-molecular cross-links of collagen molecules, respectively. Prolyl residues especially in the Y position of Gly-X-Y motif in collagen are frequently changed to a hydroxyprolyl residue by prolyl hydroxylase. Each collagen subunit has ~80–100 H
