Johannes W. Von den Hoff

Regulatory factors and cell populations involved in skeletal muscle regeneration.

Skeletal muscle regeneration is a complex process, which is not yet completely understood. Satellite cells, the skeletal muscle stem cells, become activated after trauma, proliferate, and migrate to the site of injury. Depending on the severity of the myotrauma, activated satellite cells form new multinucleated myofibers or fuse to damaged myofibers. The specific microenvironment of the satellite cells, the niche, controls their behavior. The niche contains several components that maintain satellite cells quiescence until they are activated. In addition, a great diversity of stimulatory and inhibitory growth factors such as IGF‐1 and TGF‐β1 regulate their activity. Donor‐derived satellite cells are able to improve muscle regeneration, but their migration through the muscle tissue and across endothelial layers is limited. Less than 1% of their progeny, the myoblasts, survive the first days upon intra‐muscular injection. However, a range of other multipotent muscle‐ and non‐muscle‐derived stem cells are involved in skeletal muscle regeneration. These stem cells can occupy the satellite cell niche and show great potential for the treatment of skeletal muscle injuries and diseases. The aim of this review is to discuss the niche factors, growth factors, and other stem cells, which are involved in skeletal muscle regeneration. Knowledge about the factors regulating satellite cell activity and skeletal muscle regeneration can be used to improve the treatment of muscle injuries and diseases. J. Cell. Physiol. 224:7–16, 2010

Histologic evaluation of skin-derived and collagen-based substrates implanted in palatal wounds

Tissue shortage complicates the surgery of cleft lip and palate anomalies and the healing of defects on the palate impairs growth of the dento‐alveolar complex due to scar tissue formation. Implantation of substitutes into the wound area might overcome this adverse effect. The aim of this study was to compare the tissue response to three collagen‐based (collagen type I substrate alone, or collagen coated with elastin or chondroitin‐6‐sulfate) and two skin‐derived substrates (unprocessed dermis and AlloDerm) after implantation into 12 dogs. Histology was performed at 3, 10, and 20 days postsurgery. We showed that all substrates were well tolerated. However, it is unclear whether AlloDerm was rapidly degraded or if it was sequestrated. There was no elastin or collagen present in these wounds. All collagen‐based substrates showed good epithelial regeneration, although heparan sulfate (JM 403) was absent. Wounds treated with the collagen‐based substrates contained fewer myofibroblasts at 20 days postsurgery and the type III collagen fibers in the immature scar tissue were more randomly oriented than in an untreated wound. In conclusion, palatal wounds with a dermal substrate heal with fewer indications of scar tissue formation and evoke only a mild inflammatory reaction, which is preferred over the tissue reaction in an untreated wound.

Vitamin A and clefting: putative biological mechanisms

Nutritional factors such as vitamin intake contribute to the etiology of cleft palate. Vitamin A is a regulator of embryonic development. Excess vitamin A can cause congenital malformations such as spina bifida and cleft palate. Therefore, preventive nutritional strategies are required. This review identifies putative biological mechanisms underlying the association between maternal vitamin A intake and cleft palate. Excessive vitamin A may disturb all three stages of palatogenesis: 1) during shelf outgrowth, it may decrease cell proliferation and thus prevent tissue development; 2) it may prevent shelf elevation by affecting extracellular matrix composition and hydration; and 3) during shelf fusion, it may affect epithelial differentiation and apoptosis, which precludes the formation of a continuous palate. In general, high doses of vitamin A affect palatogenesis through interference with cell proliferation and growth factors such as transforming growth factor β and platelet-derived growth factor. The effects of lower doses of vitamin A need to be investigated in greater depth in order to improve public health recommendations.

A functional model for adult stem cells in epithelial tissues

Tissue turnover, regeneration, and repair take place throughout life. Stem cells are key players in these processes. The characteristics and niches of the stem cell populations in different tissues, and even in related tissues, vary extensively. In this review, stem cell differentiation and stem cell contribution to tissue maintenance and regeneration is compared in the epithelia of the skin, the cornea, the lung, and the intestine. A hierarchical model for adult stem cells is proposed, based on the potency of stem cell subpopulations in a specific tissue. The potency is defined in terms of the maintenance, the repair, and the regeneration of the tissue. The niche supplies cues to maintain the specific stem cell potency.

Skeletal muscle fibrosis: the effect of stromal-derived factor-1α-loaded collagen scaffolds

AIM To develop a model for muscle fibrosis based on full-thickness muscle defects, and to evaluate the effects of implanted stromal-derived factor (SDF)-1α-loaded collagen scaffolds. METHODS Full-thickness defects 2 mm in diameter were made in the musculus soleus of 48 rats and either left alone or filled with SDF-1α-loaded collagen scaffolds. At 3, 10, 28 and 56 days postsurgery, muscles were analyzed for collagen deposition, satellite cells, myofibroblasts and macrophages. RESULTS A significant amount of collagen-rich fibrotic tissue was formed, which persisted over time. Increased numbers of satellite cells were present around, but not within, the wounds. Satellite cells were further upregulated in regenerating tissue when SDF-1α-loaded collagen scaffolds were implanted. The scaffolds also attracted macrophages, but collagen deposition and myofibroblast numbers were not affected. CONCLUSION Persistent muscle fibrosis is induced by full-thickness defects 2 mm in diameter. SDF-1α-loaded collagen scaffolds accelerated muscle regeneration around the wounds, but did not reduce muscle fibrosis.

The Heme-Heme Oxygenase System in Wound Healing; Implications for Scar Formation

Wound healing is an intricate process requiring the concerted action of keratinocytes, fibroblasts, endothelial cells, and macrophages. Here, we review the literature on normal wound healing and the pathological forms of wound healing, such as hypertrophic or excessive scar formation, with special emphasis on the heme-heme oxygenase (HO) system and the versatile effector molecules that are formed after HO-mediated heme degradation. Excessive scar formation following wounding is thought to relate to prolonged oxidative and inflammatory stress in the skin. Evidence is accumulating that the heme-HO system forms a novel and important target in the control of wound healing. Heme-protein derived heme can act as a potent oxidative and inflammatory stress inducer, and excess levels of heme may thus contribute to delayed resolution of oxidative and inflammatory insults in the skin. This emphasizes the need for a timely reduction of the levels of heme. Heme-binding proteins, heme transporters, and the heme degrading protein, HO, form therefore a necessary defense. Deficiencies in these defense proteins or a disturbed redox status, as in diabetic patients, may render individuals more prone to heme-induced deleterious effects. A better understanding of the heme-heme oxygenase system as target during wound healing may result in novel strategies to reduce scar formation.

FGF‐2‐loaded collagen scaffolds attract cells and blood vessels in rat oral mucosa

BACKGROUND Wound contraction and scar formation after cleft palate repair impair the growth of the maxilla. The implantation of a growth factor-loaded scaffold might solve these problems. METHODS The tissue response to fibroblast growth factor (FGF)-2 loaded collagen scaffolds was evaluated after implantation in the palate of rats. Scaffolds, with and without FGF-2, were implanted submucoperiosteally in the palate of 25 rats and evaluated after up to 16 weeks. On hematoxylin and eosin (H&E)-stained sections, the cell density and the number of giant cells within the scaffolds were quantified. Infiltration of inflammatory cells, myofibroblasts, and the number of blood vessels were quantified after immunohistochemistry. RESULTS The cell density was significantly higher in the FGF-2 group up to 4 weeks after implantation (102% at 2 weeks, P < 0.001). The number of blood vessels was also significantly higher in the FGF-2 group at 1 and 2 weeks (316% at 1 week, P = 0.003), but the myofibroblast score was lower (100% at 2 weeks, P = 0.008). A comparable mild and rapidly subsiding inflammatory response and foreign body reaction were found in both groups. CONCLUSION FGF-2-loaded scaffolds displayed a faster influx of host cells, an increased rate of vascularization, and a reduced differentiation of myofibroblasts. These scaffolds might therefore be highly suitable for intra-oral reconstructions, such as cleft palate repair.

Novel mutations in LRP6 highlight the role of WNT signaling in tooth agenesis.

Purpose:We aimed to identify a novel genetic cause of tooth agenesis (TA) and/or orofacial clefting (OFC) by combining whole-exome sequencing (WES) and targeted resequencing in a large cohort of TA and OFC patients.Methods:WES was performed in two unrelated patients: one with severe TA and OFC and another with severe TA only. After deleterious mutations were identified in a gene encoding low-density lipoprotein receptor-related protein 6 (LRP6), all its exons were resequenced with molecular inversion probes in 67 patients with TA, 1,072 patients with OFC, and 706 controls.Results:We identified a frameshift (c.4594delG, p.Cys1532fs) and a canonical splice-site mutation (c.3398-2A>C, p.?) in LRP6, respectively, in the patient with TA and OFC and in the patient with severe TA only. The targeted resequencing showed significant enrichment of unique LRP6 variants in TA patients but not in nonsyndromic OFC patients. Of the five variants in patients with TA, two affected the canonical splice site and three were missense variants; all variants segregated with the dominant phenotype, and in one case the missense mutation occurred de novo.Conclusion:Mutations in LRP6 cause TA in humans.Genet Med 18 11, 1158–1162.

Fibroblast subpopulations in intra‐oral wound healing

The objective of this study was to characterize fibroblasts at sequential time points during intra‐oral wound healing in the rat. Experimental wounds were made at several time points in the mucoperiosteum of the palate of 35‐day‐old Wistar rats. Fibroblasts were cultured from the biopsies under standard conditions for the same number of passages. The expression of the integrin subunits α1, α6, and β1; and the intermediate filaments α‐smooth muscle actin and vimentin were analyzed by flow cytometry. Western blot analysis was performed at 0, 8, and 60 days postwounding to confirm the expression of both intermediate filaments. The phenotypic profiles of fibroblasts cultured from subsequent stages in the wound healing process differed considerably. We conclude that distinct fibroblast phenotypes can be isolated from different stages in wound healing. These phenotypes remained stable during in vitro culturing. In addition, cryosections of the wound areas were made at identical time points and were immunohistochemically stained for the same antigens. The immunohistochemical staining correlated well to the flow‐cytometric data. These results suggest the occurrence of multiple subpopulations of fibroblasts with a specialized function during wound healing. We hypothesize that undesirable consequences of wound healing might be prevented through the modulation of specific fibroblast subpopulations. (WOUND REP REG 2003;11:55–63)

Palatal Wound Healing:The Effects of Scarring on Growth

Cleft palate patients often develop growth disturbances of the midfacial region after primary surgery. This is mainly caused by wound contraction and scar formation on the palate. The chapter gives an overview of the wound healing process with emphasis on wound contraction and scar formation. Some specific features of the palatal wound healing process are highlighted. Further, the effects of palatal repair on growth of the maxilla and development of the dentition are reviewed, as well as possible means to improve the clinical outcome. This review is based on clinical evaluations, experimental research in animal models, and on in vitro experiments using cell culturing and tissue engineering techniques.