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Compaction and cytoskeletal expression in GEL samples. Macroscopic images are shown (A) for P2 (left) and P3 (right) GEL samples at days 1 and 8, with cross-sectional area plotted for each day to quantitate compaction (B; mean-SEM, n = 6-15). Cytoskeletal gene expression was analyzed in day 8 samples for keratin filaments (C: KRT6A, KRT8, and KRT13), as well as other cytoskeletal proteins (D: TUBA1B, VIM, LMNA, ACTA1, and ACTA2). Values presented are mean-SEM (n = 3), with asterisks indicating p-values (*p < 0.05, **p < 0.01).
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Epimorphic regeneration in humans of complex multi-tissue structures is primarily limited to the digit tip. In a comparable mouse model, the response is level-specific in that regeneration occurs after amputation at the distal end of the terminal phalanx, but not more proximally. Recent isolation of stromal cells from CD1 murine phalangeal elements...
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... ac- cumulation, despite a low proliferation rate in P2 GEL samples, and the previous results of the scratch test in ad- herent culture, may be due to a more migratory nature of P2 cells compared to P3 cells. P2 and P3 cells are able to respond to the matrix envi- ronment in GEL culture. Qualitative images show that over 8 days the gels compacted (Fig. 8A), which is known to be due to active cellular processes, including force generation. 18 Quantification of the cross-sectional area of multiple inde- pendent samples over time (Fig. 8B) revealed that P2 cells compacted the GEL not only more quickly (by 66% vs. 22% at day 4) but also to greater levels (by 84% vs. 67% at day 8). ...
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... compared to P3 cells. P2 and P3 cells are able to respond to the matrix envi- ronment in GEL culture. Qualitative images show that over 8 days the gels compacted (Fig. 8A), which is known to be due to active cellular processes, including force generation. 18 Quantification of the cross-sectional area of multiple inde- pendent samples over time (Fig. 8B) revealed that P2 cells compacted the GEL not only more quickly (by 66% vs. 22% at day 4) but also to greater levels (by 84% vs. 67% at day 8). Considering the role of the cytoskeleton with force genera- tion, it was not unexpected that P2 cells had significantly higher gene expression levels of microtubules, nonkeratin intermediate ...
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... cells compacted the GEL not only more quickly (by 66% vs. 22% at day 4) but also to greater levels (by 84% vs. 67% at day 8). Considering the role of the cytoskeleton with force genera- tion, it was not unexpected that P2 cells had significantly higher gene expression levels of microtubules, nonkeratin intermediate filaments, and microfilaments (Fig. 8D). As was found on Fn-TCP, P3 cells did have higher levels of keratin expression (Fig. 8C); however, these proteins are not usually implicated in traction force. In addition, P2 cells had > 4-fold expression levels ( p < 0.05 for each) compared to P3 cells of extracellular matrix proteins collagen type I, collagen type IV, and ...
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... levels (by 84% vs. 67% at day 8). Considering the role of the cytoskeleton with force genera- tion, it was not unexpected that P2 cells had significantly higher gene expression levels of microtubules, nonkeratin intermediate filaments, and microfilaments (Fig. 8D). As was found on Fn-TCP, P3 cells did have higher levels of keratin expression (Fig. 8C); however, these proteins are not usually implicated in traction force. In addition, P2 cells had > 4-fold expression levels ( p < 0.05 for each) compared to P3 cells of extracellular matrix proteins collagen type I, collagen type IV, and fibronectin ( Fig. 9). Thus, in an exogenously matrix- rich environment, such as collagen type I ...
Citations
... 19 adult stem cells grafted into a fetal extracellular matrix (ECM) regain their "stemness youth," while fetal stem cells when placed in adult ECM did not bring quali-quantitatively improvements, adapting to the environment in which they were immersed. 20,21 Similar behavior is observed the senescent fibroblasts cultured in young ECM. The mechanisms related to these "rejuvenation" capabilities are unknown, although it is suggested to depend on the telomeres. ...
BACKGROUND: Reconstructive surgery is one of the most challenging features of the surgical defects and scar is the main answer to wound for re-gaining the tissue continuity, sometimes however resulting on a not functionally and aesthetically complete success. Bioscaffolds are gaining more and more interest in medicine due to their biologic and functional properties and Matriderm® is a bovine dermic bioscaffold conceived for orthopaedic, plastic and dermatologic purposes. The aim of this clinical study is to show the advantages of the use of Matriderm® in different situations of oral surgery.
METHODS: Forty patients with need of anatomic ablation in oral region and with a wound healing process by second intention, mucosal graft or local flaps were randomly selected. In all the cases, a fragment of Matriderm® was grafted into the surgical defect and fixed by a rapid reabsorption suture.
RESULTS: All patients treated showed a complete restitutio ad integrum, no early or late bleeding phenomena were found and the healing time was very shorter when compared to the traditional surgery.
CONCLUSIONS: Use of Matriderm® in oral surgery may be considered a good approach to reduce the healing time, to improve the final result and to avoid the risk of recurrences.
KEY WORDS: Bioscaffold - Wound healing - Mucosal substitute - Oral surgery - Tonsillectomy
... Protein Expression. Standard protocols for immunohistochemistry and flow cytometry were used to determine protein expression as described previously 12,48 . EBs were fixed in 4% formaldehyde, paraffin embedded, and sectioned (6 μ m slices). ...
The cytoskeletal filament vimentin is inherent to the endothelial phenotype and is critical for the proper function of endothelial cells in adult mice. It is unclear, however, if the presence of vimentin is necessary during differentiation to the endothelial phenotype. Here we evaluated gene and protein expression of differentiating wild type embryonic stem cells (WT ESCs) and vimentin knockout embryonic stem cells (VIM −/− ESCs) using embryoid bodies (EBs) formed from both cell types. Over seven days of differentiation VIM −/− EBs had altered morphology compared to WT EBs, with a rippled outer surface and a smaller size due to decreased proliferation. Gene expression of pluripotency markers decreased similarly for EBs of both cell types; however, VIM −/− EBs had impaired differentiation towards the endothelial phenotype. This was quantified with decreased expression of markers along the specification pathway, specifically the early mesodermal marker Brachy-T, the lateral plate mesodermal marker FLK1, and the endothelial-specific markers TIE2, PECAM, and VE-CADHERIN. Taken together, these results indicate that the absence of vimentin impairs spontaneous differentiation of ESCs to the endothelial phenotype in vitro.
... 13,15,91 Connective tissue fibroblasts isolated 97 from regeneration-competent regions also had higher proliferation rates than those isolated from regeneration-incompetent regions upon in vitro culture under multiple two-dimensional and three-dimensional culture conditions (adherent, suspension, and collagen gels). 98 Proliferative differences in the in vitro expanded cells were retained even after reimplantation into amputated joints. 97 Thus, those cells that participate in the formation of a blastema-like structure seem to have a greater inherent proliferative capacity compared to analogous cells from nonregenerative regions. ...
Approximately 2 million people have had limb amputations in the United States due to disease or injury, with more than 185,000 new amputations every year. The ability to promote epimorphic regeneration, or the regrowth of a biologically based digit or limb, would radically change the prognosis for amputees. This ambitious goal includes the regrowth of a large number of tissues that need to be properly assembled and patterned to create a fully functional structure. We have yet to even identify, let alone address, all the obstacles along the extended progression that limit epimorphic regeneration in humans. This review aims to present introductory fundamentals in epimorphic regeneration to facilitate design and conduct of research from a tissue engineering and regenerative medicine perspective. We describe the clinical scenario of human digit healing, featuring published reports of regenerative potential. We then broadly delineate the processes of epimorphic regeneration in non-mammalian systems and describe a few mammalian regeneration models. We give particular focus to the murine digit tip, which allows for comparative studies of regeneration-competent and regeneration-incompetent outcomes in the same animal. Finally, we describe a few forward-thinking opportunities for promoting epimorphic regeneration in humans.
... These studies provide evidence that connective tissue fibroblasts possess and maintain stable position-specific characteristics, and that these cells do not inherently dictate whether or not a regenerative response is induced at an amputation wound. Other in vitro studies using P2/P3 cell lines suggest unique differences in how these cells can modify the physical microenvironment of the blastema and, as well, initiate inductive signaling pathways known to be critical for a regenerative response (Lynch & Ahsan 2013, 2014. ...
In the mouse, digit tip regeneration progresses through a series of discreet stages that include inflammation, histolysis, epidermal closure, blastema formation and redifferentiation. Recent studies reveal how each regenerative stage influences subsequent stages to establish a blastema that directs the successful regeneration of a complex mammalian structure. The focus of this review is on early events of healing and how an amputation wound transitions into a functional blastema. The stepwise formation of a mammalian blastema is proposed to provide a model for how specific targeted treatments can enhance regenerative performance in humans.This article is protected by copyright. All rights reserved.
... 11 Differences in cell proliferation and migration were also evident in in vitro studies, however, where P3 cells were significantly more proliferative than P2 cells for a number of two-and three-dimensional microenvironments. 12 The mechanisms that regulate these differences in proliferative and migratory capacity, processes necessary for the formation of a stable cell mass during the initial stages of regeneration, are still unknown. ...
... The effects of fibroblasts on P3 cells were similarly evaluated as for P2 cells. Consistent with previous publications 11,12 the inherent proliferative rate of P3 cells is markedly higher than that of P2 cells (3-6 · greater in cell number for these studies). Nonetheless, proliferation of P3 cells, unlike that of P2 cells, was not significantly ( p = 0.6) affected by the presence of neonatal or adult fibroblasts in indirect co-culture (Fig. 1C left). ...
... Previously, we had shown that P2 and P3 cells differ greatly with regard to proliferation and migration: P3 cells are more proliferative, while P2 cells are more migratory. 12 During regeneration in vivo, adjacent cell phenotypes may modulate these two processes by secretion of specific growth factors. In these in vitro studies, we showed that signaling from fibroblasts, in particular, can considerably adjust the proliferation and migration of P2 and P3 cells. ...
The capacity to regenerate complex tissue structures after amputation in humans is limited to the digit tip. In a comparable mouse digit model, which includes both distal regeneration-competent and proximal regeneration-incompetent regions, successful regeneration involves precise orchestration of complex microenvironmental cues including paracrine signaling via heterogeneous cell-cell interactions. Initial cellular processes, such as proliferation and migration, are critical in the formation of an initial stable cell mass and the ultimate regenerative outcome. Hence, the objective of these in vitro studies was to investigate the effect of soluble factors secreted by fibroblasts and mesenchymal stem cells (MSCs) on the proliferation and migration of cells from the regeneration-competent (P3) and -incompetent (P2) regions of the mouse digit tip. We found that P2 and P3 cells were more responsive to fibroblasts than MSCs and that the effects were mediated by bi-directional communication. To initiate understanding of the specific soluble factors that may be involved in the fibroblast-mediated changes in migration of P2 and P3 cells, bone morphogenic protein 2 (BMP2) was exogenously added to the medium. We found that changes in migration of P3 cells were similar when exposed to BMP2 or co-cultured with fibroblasts, indicating that BMP signaling may be responsible for the migratory response of P3 cells to the presence of fibroblasts. Furthermore, BMP2 expression in fibroblasts was shown to be responsive to tensile strain, as is present during wound closure. Therefore, these in vitro studies indicate that regenerative processes may be regulated by fibroblast-secreted soluble factors, which in turn are modulated by both crosstalk between heterogeneous phenotypes and the physical microenvironment of the healing site.
Despite approaches in regenerative medicine using stem cells, bio-engineered scaffolds, and targeted drug delivery to enhance human tissue repair, clinicians remain unable to regenerate large-scale, multi-tissue defects in situ. The study of regenerative biology using mammalian models of complex tissue regeneration offers an opportunity to discover key factors that stimulate a regenerative rather than fibrotic response to injury. For example, although primates and rodents can regenerate their distal digit tips, they heal more proximal amputations with scar tissue. Rabbits and African spiny mice re-grow tissue to fill large musculoskeletal defects through their ear pinna, while other mammals fail to regenerate identical defects and instead heal ear holes through fibrotic repair. This Review explores the utility of these comparative healing models using the spiny mouse ear pinna and the mouse digit tip to consider how mechanistic insight into reparative regeneration might serve to advance regenerative medicine. Specifically, we consider how inflammation and immunity, extracellular matrix composition, and controlled cell proliferation intersect to establish a pro-regenerative microenvironment in response to injuries. Understanding how some mammals naturally regenerate complex tissue can provide a blueprint for how we might manipulate the injury microenvironment to enhance regenerative abilities in humans. Stem Cells Translational Medicine 2017.
The regrowth of amputated digit tips represents a unique regenerative healing in mammals with subcutaneous volume regrowth, restoration of dactylogram, and suppression of scar formation. Although factor analysis in amphibians and even in mice is easy to obtain, safety of harvesting biomaterial from human digit tip amputations for analysis has not yet been described.
The aim of this study was to evaluate if recovering wound exudate does hamper clinical outcome or influence microbiologic or inflammation status.
A predefined cohort of 18 patients with fresh digit tip amputations was randomly assigned to receive standard therapy (debridement, occlusive dressing) with (n = 9) or without (n = 9) collection of the whole wound exudate in every dressing change. Primary endpoint (lengthening) and secondary endpoints (regeneration of dactylogram, nail bed and bone healing, time to complete wound closure, scar formation, 2-point discrimination, microbiologic analysis, inflammatory factors interleukin (IL)-1α, tumor necrosis factor-α, IL-4, and IL-6) were determined by an independent, blinded observer.
Patients’ characteristics showed no significant differences between the groups. All patients completed the study to the end of 3 months follow-up. Exudate collection did not influence primary and secondary endpoints. Furthermore, positive microbiologic findings as well as pus- and necrosis-like appearance neither impaired tissue restoration nor influenced inflammatory factor release.
Here, the authors developed an easy and safe protocol for harvesting wound exudate from human digit tip amputations. For the first time, it was shown that harvesting does not impair regenerative healing. Using this method, further studies can be conducted to analyze regeneration associated factors in the human digit tip.
DRKS.de Identifier: DRKS00006882 (UTN: U1111-1166-5723).
