Platelet‐derived transforming growth factor‐β1 promotes keratinocyte proliferation in cutaneous wound healing

Platelets are a recognised potent source of transforming growth factor‐β1 (TGFβ1), a cytokine known to promote wound healing and regeneration by stimulating dermal fibroblast proliferation and extracellular matrix deposition. Platelet lysate has been advocated as a novel personalised therapeutic to treat persistent wounds, although the precise platelet‐derived growth factors responsible for these beneficial effects have not been fully elucidated. The aim of this study was to investigate the specific role of platelet‐derived TGFβ1 in cutaneous wound healing. Using a transgenic mouse with a targeted deletion of TGFβ1 in megakaryocytes and platelets (TGFβ1^fl/fl.PF4‐Cre), we show for the first time that platelet‐derived TGFβ1 contributes to epidermal and dermal thickening and cellular turnover after excisional skin wounding. In vitro studies demonstrate that human dermal fibroblasts stimulated with platelet lysate containing high levels of platelet‐derived TGFβ1 did not exhibit enhanced collagen deposition or proliferation, suggesting that platelet‐derived TGFβ1 is not a key promoter of these wound healing processes. Interestingly, human keratinocytes displayed enhanced TGFβ1‐driven proliferation in response to platelet lysate, reminiscent of our in vivo findings. In summary, our novel findings define and emphasise an important role of platelet‐derived TGFβ1 in epidermal remodelling and regeneration processes during cutaneous wound healing.     

重组人表皮生长因子和碱性成纤维生长因子促进人角膜上皮细胞的增殖

背景：重组人表皮生长因子和碱性成纤维生长因子的制剂临床上已经用于眼表创伤的修复,但是对于使用何种浓度的生长因子就能够最大程度的促进愈合以及两种生长因子促进创面愈合的效果比较一直存在争议。 目的：初步观察重组人表皮生长因子、碱性成纤维生长因子对培养的人角膜上皮细胞克隆的影响。 方法：用不同浓度的重组人表皮生长因子和碱性成纤维生长因子作用于体外培养的人角膜上皮细胞,应用四甲基偶氮唑盐比色法分别在不同浓度的生长因子作用人角膜上皮细胞3,5,7 d后检测人角膜上皮细胞的增殖能力;平板克隆形成实验法观察细胞克隆形态及计算细胞克隆形成率。 结果与结论：不同质量浓度的重组人表皮生长因子和碱性成纤维生长因子分别对人角膜上皮细胞干预5d后,重组人表皮生长因子和碱性成纤维生长因子在10μg/L质量浓度时MTT值最大;质量浓度10μg/L重组人表皮生长因子促进人角膜上皮细胞克隆形成率高于质量浓度10μg/L碱性成纤维生长因子（P=0.02）。结果证实,重组人表皮生长因子和碱性成纤维生长因子均能促进人角膜上皮细胞增殖并增加其克隆形成能力。质量浓度10μg/L 重组人表皮生长因子干预5 d促进人角膜上皮细胞克隆形成效果最好。     

Amniotic membrane stimulates cell migration by modulating transforming growth factor‐β signalling

Keratinocyte migration is a mandatory aspect of wound healing. We have previously shown that amniotic membrane (AM) applied to chronic wounds assists healing through a process resulting in the overexpression of c‐Jun at the wound's leading edge. We have also demonstrated that AM modifies the genetic programme induced by transforming growth factor‐ß (TGF‐ß) in chronic wounds. Here we used a scratch assay of mink lung epithelial cells (Mv1Lu) and a spontaneously immortalized human keratinocyte cell line (HaCaT) cells to examine the influence of AM application on the underlying signalling during scratch closure. AM application induced c‐Jun phosphorylation at the leading edge of scratch wounds in a process dependent on MAPK and JNK signalling. Strikingly, when the TGF‐ß‐dependent Smad‐activation inhibitor SB431542 was used together with AM, migration improvement was partially restrained, whereas the addition of TGF‐ß had a synergistic effect on the AM‐induced cell migration. Moreover, antagonizing TGF‐ß with specific antibodies in both cell lines or knocking out TGF‐ß receptors in Mv1Lu cells had similar effects on cell migration as using SB431542. Furthermore, we found that AM was able to attenuate TGF‐ß‐Smad signalling specifically at the migrating edge; AM treatment abated Smad2 and Smad3 nuclear localization in response to TGF‐ß in a process dependent on mitogen‐activated protein kinase kinase 1 (MEK1) activation but independent of EGF receptor or JNK activation. The involvement of Smad signalling on AM effects on HaCaT keratinocytes was further corroborated by overexpression of either Smad2 or Smad3 and the use of Smad phosphorylation‐specific inhibitors, revealing a differential influence on AM‐induced migration for each Smad. Thus, AM TGF‐ß‐Smad signalling abating is essential for optimal cell migration and wound closure.     

转化生长因子-β、碱性成纤维生长因子和血小板衍生生长因子在骨折愈合中的表达

目的:观察转化生长因子-β(TGF-β)、碱性成纤维细胞生长因子(bFGF)和血小板衍生生长因子(PDGF)在骨折愈合中的表达和分布情况,进而探讨其作用机制。方法:选用SD大鼠制作胫骨骨折愈合模型,伤后不同时期处死取材,分别进行组织学和TGF-β、bFGF和PDGF免疫组化染色观察。结果:(1)伤后3d开始形成原始骨痂。1周时肉芽组织中的间质细胞开始分化为软骨细胞,软骨形成后再进行软骨内化骨。4周时形成连接骨折端的桥接骨痂。(2)伤后早期血肿中炎性细胞表达bFGF、PDGF。伤后1周骨膜增殖细胞、肉芽组织中的成纤维细胞、内皮细胞、骨端骨细胞以及原始骨痂成骨细胞表达TGF-β、bFGF和PDGF。伤后2周软骨细胞表达TGF-β、bFGF和PDGF。结论:TGF-β、bFGF和PDGF有着各自的表达和分布特点,并共同调解骨原细胞的增殖和成骨细胞、软骨细胞的分化,最终完成骨折愈合。     

The influence of sustained dual‐factor presentation on the expansion and differentiation of neural progenitors in affinity‐binding alginate scaffolds

Biomaterials capable of controlling the release of multiple growth factors (GFs) could potentially promote the integration of co‐transplanted neural progenitor cells (NPCs) and stimulate the plasticity and regenerability of the lesioned spinal cord. As a first step towards the employment of such a vehicle for cell therapy, this study examined the capability of an alginate–sulphate/alginate scaffold, able to capture and rigorously control the release of GFs, to promote the expansion and lineage differentiation of NPCs in vitro. Epidermal growth factor (EGF) and fibroblast growth factor‐2 (bFGF) were affinity‐bound to alginate–sulphate (200 ng/scaffold) and the bioconjugates were mixed with partially calcium‐crosslinked alginate. NPCs isolated from 18 day‐old rat embryo brains and seeded into the scaffold during preparation were found to proliferate and differentiate within the vehicle. A continuous release of both bFGF and EGF was noted for a period of 21 days. The concentrations of released GFs were sufficient to promote extensive NPC proliferation at initial cultivation times; the number of neurospheres in the scaffold was twice the number found in the 2D cultures supplemented with 20 ng/ml each factor every 3 days. Between days 10–14, when the GF concentrations had substantially declined, extensive cell migration from the neurospheres as well as lineage differentiation were noted in the scaffold; immunocytochemical analyses confirmed the presence of neurons, astrocytes and oligodendrocytes.The scaffold has a potential to serve as cell delivery vehicle, with proven capability to promote cell retention and expansion, while enabling NPC lineage differentiation in situ. Copyright © 2013 John Wiley & Sons, Ltd.     

TGF‐β1 enhances contractility in engineered skeletal muscle

Scaffoldless engineered 3D skeletal muscle tissue created from satellite cells offers the potential to replace muscle tissue that is lost due to severe trauma or disease. Transforming growth factor‐beta 1 (TGF‐β1) plays a vital role in mediating migration and differentiation of satellite cells during the early stages of muscle development. Additionally, TGF‐β1 promotes collagen type I synthesis in the extracellular matrix (ECM) of skeletal muscle, which provides a passive elastic substrate to support myofibres and facilitate the transmission of force. To determine the role of TGF‐β1 in skeletal muscle construct formation and contractile function in vitro, we created tissue‐engineered 3D skeletal muscle constructs with varying levels of recombinant TGF‐β1 added to the cell culture medium. Prior to the addition of TGF‐β1, the primary cell population was composed of 75% Pax7‐positive cells. The peak force for twitch, tetanus and spontaneous force were significantly increased in the presence of 2.0 ng/ml TGF‐β1 when compared to 0, 0.5 and 1.0 ng/ml TGF‐β1. Visualization of the cellular structure with H&E and with immunofluorescence staining for sarcomeric myosin heavy chains and collagen type I showed denser regions of better organized myofibres in the presence of 2.0 ng/ml TGF‐β1 versus 0, 0.5 and 1.0 ng/ml. The addition of 2.0 ng/ml TGF‐β1 to the culture medium of engineered 3D skeletal muscle constructs enhanced contractility and extracellular matrix organization. Copyright © 2012 John Wiley & Sons, Ltd.     

TGF‐β1‐activated type 2 dendritic cells promote wound healing and induce fibroblasts to express tenascin c following corneal full‐thickness hydrogel transplantation

We showed previously that 1‐ethyl‐3‐(3‐dimethylamino‐propyl)‐carbodiimide hydrochloride (EDC) cross‐linked recombinant human collagen III hydrogels promoted stable regeneration of the human cornea (continued nerve and stromal cell repopulation) for over 4 years. However, as EDC cross linking kinetics were difficult to control, we additionally tested a sterically bulky carbodiimide. Here, we compared the effects of two carbodiimide cross linkers—bulky, aromatic N‐cyclohexyl‐N0‐(2‐morpholinoethyl)‐carbodiimide (CMC), and nonbulky EDC—in a mouse corneal graft model. Murine corneas undergoing full‐thickness implantation with these gels became opaque due to dense retro‐corneal membranes (RCM). Corneal epithelial cytokeratin 12 and alpha smooth muscle actin indicative of functional tissue regeneration and wound contraction were observed in RCM surrounding both hydrogel types. However, quantitatively different levels of infiltrating CD11c⁺ dendritic cells (DC) were found, suggesting a hydrogel‐specific innate immune response. More DC infiltrated the stroma surrounding EDC‐N‐hydroxysuccinimide (NHS) hydrogels concurrently with higher fibrosis‐associated tenascin c expression. The opposite was true for CMC‐NHS gels that had previously been shown to be more tolerising to DC. In vitro studies showed that DC cultured with transforming growth factor β1 (TGF‐β1) induced fibroblasts to secrete more tenascin c than those cultured with lipopolysaccharide and this effect was blocked by TGF‐β1 neutralisation. Furthermore, tenascin c staining was found in 40‐ to 50μm long membrane nanotubes formed in fibroblast/DC cocultures. We suggest that TGF‐β1 alternatively activated (tolerising) DC regulate fibroblast‐mediated tenascin c secretion, possibly via local production of TGF‐β1 in early wound contraction, and that this is indirectly modulated by different hydrogel chemistries.     

Mesenchymal stem cells can be recruited to wounded tissue via hepatocyte growth factor‐loaded biomaterials

Mesenchymal stem cells (MSC) are precursor cells of mesodermal tissue and, because of their trophic phenotype, they are known to play beneficial roles in wound healing. In addition, various tissue engineering strategies are based on MSC/biomaterial constructs. As the isolation and expansion of MSCs is a long‐term process, a major goal is to develop an endogenous stem cell recruitment system that circumvents all ex vivo steps generally used for tissue engineering. Therefore collagen and silk fibroin were loaded with hepatocyte growth factor (HGF), a chemoattractant for MSCs. Collagen was mixed with HGF during polymerization, while silk fibroin and HGF were produced as fusion proteins by transgenic silkworms. To demonstrate release of active HGF, enzyme‐linked immunosorbent assay, in vitro migration assays and animal studies were performed to demonstrate MSC migration in vivo, followed by detailed examinations of the immunological effects of the biomaterials. Hepatocyte growth factor was released burst‐like, both from silk fibroin and collagen during the first 8 h and gradually for up to 168 h in vitro. Directed migration in vitro was demonstrated when MSCs were exposed to HGF. In vivo, HGF‐loaded collagen and silk fibroin were tolerated as subcutaneous implants. In addition, it was proved that endogenous MSCs were recruited from the local environment. These results show for the first time recruitment of endogenous MSCs to HGF‐loaded collagen (fast degradable) and silk fibroin scaffolds (long‐term degradable) in vitro and in vivo. This knowledge could be applied to make off‐the‐shelf, readily available constructs for use in patients with chronic wound or burns. Copyright © 2016 John Wiley & Sons, Ltd.     

Facial nerve regeneration using basic fibroblast growth factor‐impregnated gelatin microspheres in a rat model

Basic fibroblast growth factor (bFGF) plays a crucial role in the regeneration of peripheral nerve defects by affecting nerve cells, Schwann cells and fibroblasts, and by promoting axon outgrowth from the proximal nerve stump. However, the use of exogenous bFGF for in vivo regeneration of the peripheral nerves is limited by its short in vivo half‐life. In this study, a drug delivery system for bFGF was developed that uses acidic gelatin hydrogel, which sustainably released bFGF in vivo over several weeks; its ability to promote peripheral nerve regeneration was also examined. In 8‐week‐old Lewis rats, 7‐mm gaps were made in the buccal branch of the left facial nerve. Acidic gelatin hydrogel microspheres (10 µl) with or without bFGF (50 µg) were infused into a 10 mm silicone tube using a micropipette, and the silicone tube was then implanted into the gap. A 1‐mm long nerve stump was inserted into each end of the tube. Histological examination at 7 weeks after implantation revealed (1) a significantly increased rate of nerve regeneration, (2) inducement of a number of regenerating nerve axons, and (3) a better degree of maturation of nerve axons in the bFGF microsphere group than that in the bFGF‐free microsphere group. Copyright © 2014 John Wiley & Sons, Ltd.     

A virally inactivated platelet‐derived growth factor/vascular endothelial growth factor concentrate fractionated from human platelets

BACKGROUND: Human platelet concentrates (PCs) may be a source material to produce purified growth factors (GFs) for clinical use or cell therapy. However, no fractionation process of therapeutic‐grade GF from PCs has ever been developed. STUDY DESIGN AND METHODS: PCs were virally inactivated by solvent/detergent (S/D) treatment, subjected to oil extraction to remove part of the S/D agents, and fractionated on a SP‐Sepharose (SP) chromatographic column equilibrated in a phosphate‐buffered saline (PBS) buffer, pH 7.5. The breakthrough was recovered, and the column was washed with the PBS buffer and then eluted by a 0.7 mol/L NaCl‐PBS buffer pH 7.5 (SP‐eluate). The SP‐breakthrough and SP‐eluate were characterized for their content in GF, proteins, lipids, and S/D agents. The MTS value of three cell lines cultivated in a medium containing 10% fetal bovine serum supplemented with 1% to 3% of SP‐eluate or recombinant human (rHu) platelet‐derived growth factor (PDGF)‐BB was compared. RESULTS: The SP‐eluate contained a mean of 47, 17, and 6 ng/mL PDGF‐AB, ‐BB, and ‐AA, respectively, and 0.26 ng/mL vascular endothelial growth factor (VEGF). It was largely depleted of transforming growth factor‐β1 (2.33 ng/mL), epidermal growth factor (0.09 ng/mL), insulin‐like growth factor (3.40 ng/mL), albumin, immunoglobulin (Ig)G, IgM, IgA, and fibrinogen, which were mostly in the breakthrough. tri‐n‐butyl phosphate and Triton X‐45 were less than 2 ppm. Cell growth–promoting activity of the SP‐eluate was at least as good as that of rHu‐PDGF‐BB. CONCLUSION: Human PC can be fractionated into a purified, virally inactivated PDGF and VEGF concentrate, opening perspectives for the development of a new range of blood products for clinical use and cell therapy procedures.     

Effect of double growth factor release on cartilage tissue engineering

The effects of double release of insulin‐like growth factor I (IGF‐I) and growth factor β1 (TGF–β1) from nanoparticles on the growth of bone marrow mesenchymal stem cells and their differentiation into cartilage cells were studied on PLGA scaffolds. The release was achieved by using nanoparticles of poly(lactic acid‐co‐glycolic acid) (PLGA) and poly(N‐isopropylacrylamide) (PNIPAM) carrying IGF‐I and TGF–β1, respectively. On tissue culture polystyrene (TCPS), TGF‐β1 released from PNIPAM nanoparticles was found to have a significant effect on proliferation, while IGF‐I encouraged differentiation, as shown by collagen type II deposition. The study was then conducted on macroporous (pore size 200–400 µm) PLGA scaffolds. It was observed that the combination of IGF‐I and TGF‐β1 yielded better results in terms of collagen type II and aggrecan expression than GF‐free and single GF‐containing applications. It thus appears that gradual release of a combination of growth factors from nanoparticles could make a significant contribution to the quality of the engineered cartilage tissue. Copyright © 2011 John Wiley & Sons, Ltd.     

Enhanced intestinal anastomotic healing with gelatin hydrogel incorporating basic fibroblast growth factor

Anastomotic leakage is a common complication of intestinal surgery. In an attempt to resolve this issue, a promising approach is enhancement of anastomotic wound healing. A method for controlled release of basic fibroblast growth factor (bFGF) using a gelatin hydrogel was developed with the objective of investigating the effects of this technology on intestinal anastomotic healing. The small intestine of Wistar rats was cut, end‐to‐end anastomosis was performed and rats were divided into three groups: bFGF group (anastomosis wrapped with a hydrogel sheet incorporating bFGF), PBS group (wrapped with a sheet incorporating phosphate‐buffered saline solution) and NT group (no additional treatment). Degradation profiles of gelatin hydrogels in vivo and histological examinations were performed using gelatin hydrogels with various water contents and bFGF concentrations to define the optimal bFGF dose and hydrogel biodegradability. The anastomotic wound healing process was evaluated by histological examinations, adhesion‐related score and bursting pressure. The optimal water content of the hydrogel and bFGF dose was determined as 96% and 30 µg per sheet, respectively. Application of bFGF significantly enhanced neovascularization, fibroblast infiltration and collagen production around the anastomotic site when compared with the other groups. Bursting pressure was significantly increased in the bFGF group. No significant difference was observed in the adhesion‐related score among the groups and no anastomotic obstruction and leakage were observed. Therefore controlled release of bFGF enhanced healing of an intestinal anastomosis during the early postoperative period and is a promising method to suppress anastomotic leakage. Copyright © 2013 John Wiley & Sons, Ltd.     

碱性成纤维细胞生长因子及血小板源性生长因子在人股骨颈骨折中的表达及相互关系

目的 研究股骨颈骨折后不同时期碱性成纤维细胞生长因子(bFGF)及血小板源性生长因子(PDGF)的表达及分布特点.方法 应用免疫组化PV超敏法对36例人股骨颈骨折标本中bFGF、PDGF蛋白进行检测,应用CMIAS真彩色医学图像分析系统对各组免疫组化结果进行吸光度(A)检测及分析,同时对bFGF、PDGF表达进行相关性分析.结果 ①伤后1周组9例,bFGF蛋白在骨折处间充质细胞、单核细胞、血管内皮细胞表达最为显著,A为(0.4076 ±0.0902);PDGF蛋白在间充质细胞内呈弱阳性表达,而在血管内皮细胞内呈强阳性表达,A为(0.2261 ±0.0636).伤后2周组9例,bFGF、PDGF表达于成纤维细胞、血管内皮细胞、新生的软骨细胞及基质、成骨细胞中,A分别为(0.6404±0.0920)和(0.7457±0.0756),与伤后1周比较,差异有统计学意义(P＜0.05,P＜0.01).伤后3周组9例,bFGF及PDGF蛋白表达的OD分别为(0.7168 ±0.1346)和(0.8033.±0.0491),与伤后2周相比差异无统计学意义(P均＞0.05).伤后4周组9例,成熟的骨组织、软骨组织中bFGF、PDGF蛋白均呈弱阳性或阴性反应,但幼稚骨组织、软骨组织中则呈阳性表达,OD分别为(0.5374 ±0.1210)和(0.7068±0.1255),但表达程度低于伤后3周(P均＜0.05).②股骨颈骨折后1、2、3、4周bFGF与PDGF蛋白表达均成正相关(r分别为0.792、0.834、0.880、0.766,P均＜0.05).结论 ①bFGF在骨折愈合早期能诱导间充质细胞、成骨细胞、软骨细胞及血管内皮等细胞的分裂、增殖,促进新生血管及新骨的生成.②PDGF是骨细胞调节剂,可刺激软骨细胞和成骨细胞的增殖、分化并可间接地诱导血管内皮细胞增殖与新生血管生成.③bFGF及PDGF均为骨性生长因子,二者互相促进,共同调节成骨细胞及血管增殖和分化,最终完成骨折修复过程.     

Comparison of growth factor and interleukin content of adult peripheral blood and cord blood serum eye drops for cornea and ocular surface diseases

Introduction

Various blood-derived products have been proposed for the topical treatment of ocular surface diseases. The aim of the study was to compare the different content of Growth Factors (GFs) and Interleukins (ILs) in peripheral blood (PB-S) and Cord Blood (CB-S) sera.

Platelet‐activated serum might have a therapeutic effect on damaged articular cartilage

Platelet‐activated serum (PAS) was collected from rabbits. This contains high concentrations of growth factors, including vascular endothelial growth factor (VEGF), platelet‐derived growth factor (PDGF)‐BB, and transforming growth factor‐beta (TGF‐β). PAS was injected into the knee joints of Japanese White rabbits subjected to anterior cruciate ligament transection (ACL‐T) to investigate its therapeutic effects on articular cartilage. The effect of Avastin (an anti‐VEGF monoclonal antibody) on VEGF expression was also investigated. The levels of VEGF, PDGF‐BB, and TGF‐β in PAS, platelet‐rich plasma (PRP) and autologous serum from untreated rabbits were analysed by enzyme‐linked immunosorbent assays. The samples (n = 24 rabbits) were divided into control (C), PAS (S), Avastin (A) and PAS + Avastin (S + A) treatment groups. Intra‐articular injections were administered weekly for 7 weeks after ACL‐T, during which the weight distribution ratios of the damaged limbs were evaluated. Histological evaluation was performed 12 weeks after ACL‐T using Mankin score. The VEGF, PDGF‐BB and TGF‐β expression levels were significantly higher (P < 0.05) in the PAS than in the PRP or autologous serum samples. The weight distribution ratios of damaged limbs improved significantly after ACL‐T in all treatment groups (P < 0.05). The proximal medial, distal medial and lateral aspects of joints in the treatment groups showed significant differences in Mankin scores compared with controls (P < 0.05). The damaged limb weight distribution ratios, Mankin scores and articular cartilage structure did not differ significantly among the three treatment groups, which all showed significant improvements in structure compared with controls. Copyright © 2017 John Wiley & Sons, Ltd.     

Repair of an osteochondral defect by sustained delivery of BMP‐2 or TGFβ1 from a bilayered alginate–PLGA scaffold

Regeneration of cartilage defects can be accelerated by localized delivery of appropriate growth factors (GFs) from scaffolds. In the present study we analysed the in vitro and in vivo release rates and delivery efficacies of transforming growth factor‐β1 (TGFβ1) and bone morphogenetic protein‐2 (BMP‐2) from a bilayered system, applied for osteochondral defect repair in a rabbit model. A bone‐orientated, porous PLGA cylinder was overlaid with GF containing PLGA microspheres, dispersed in an alginate matrix. Four microsphere formulations were incorporated: (a) blank ones; (b) microspheres containing 50 ng TGFβ1; (c) microspheres containing 2.5 µg BMP‐2; and (d) microspheres containing 5 µg BMP‐2. Release kinetics and tissue distributions were determined using iodinated (¹²⁵I) GFs. Bioactivity of in vitro released BMP‐2 and TGFβ1 was confirmed in cell‐based assays. In vivo release profiles indicated good GF release control. 20% of BMP‐2 and 15% of TGFβ1 were released during the first day. Virtually the total dose was delivered at the end of week 6. Significant histological differences were observed between untreated and GF‐treated specimens, there being especially relevant short‐term outcomes with 50 ng TGFβ1 and 5 µg BMP‐2. Although the evaluation scores for the newly formed cartilage did not differ significantly, 5 µg BMP‐2 gave rise to higher quality cartilage with improved surface regularity, tissue integration and increased collagen‐type II and aggrecan immunoreactivity 2 weeks post‐implantation. Hence, the bilayered system controlled GF release rates and led to preserved cartilage integrity from 12 weeks up to at least 24 weeks. Copyright © 2012 John Wiley & Sons, Ltd.     

Collagen sponge scaffolds containing growth factors for the functional regeneration of tracheal epithelium

Tracheal epithelia have barrier and mucociliary clearance functions that prevent invasion of extraneous particles and infectious materials. Hence, following tracheal reconstructions, functional and morphological regeneration of epithelia is required to prevent respiratory declines and infectious diseases. Although growth factors (GFs) promote the regeneration of tracheal epithelial morphologies, it remains unclear whether tracheal grafts containing GFs are beneficial for regeneration of tracheal epithelial functions. Thus, we fabricated collagen sponge scaffolds containing insulin‐like GF‐1 (IGF‐1) and the basic fibroblast, hepatocyte, and epidermal GFs (bFGFs, HGFs, and EGFs, respectively), and we evaluated the effects of the grafts on the functional regeneration of tracheal epithelia. Partial tracheal defects were imposed surgically, and collagen sponges containing IGF‐1, bFGF, HGF, or EGF were then transplanted to defect sites. Subsequent immunofluorescence studies suggested that EGF and bFGF contribute to regular distributions of tight junction molecules, and tracer permeability assays suggested that EGF and bFGF promote regeneration of barrier function. Increased ciliogenesis was also observed using scanning electron microscopy in reconstructed regions treated with EGF‐ and bFGF‐supplemented collagen sponges. However, bFGF‐supplemented collagen sponges led to greater microsphere transport than did EGF‐supplemented sponges. The present data suggested that collagen sponge scaffold containing bFGF promotes functional regeneration of tracheal epithelial tissues.     

Human adipose tissue‐derived tenomodulin positive subpopulation of stem cells: A promising source of tendon progenitor cells

Cell‐based therapies are of particular interest for tendon and ligament regeneration given the low regenerative potential of these tissues. Adipose tissue is an abundant source of stem cells, which may be employed for the healing of tendon lesions. However, human adult multipotent adipose‐derived stem cells (hASCs) isolated from the stromal vascular fraction of adipose tissue originate highly heterogeneous cell populations that hinder their use in specific tissue‐oriented applications. In this study, distinct subpopulations of hASCs were immunomagnetic separated and their tenogenic differentiation capacity evaluated in the presence of several growth factors (GFs), namely endothelial GF, basic‐fibroblast GF, transforming GF‐β1 and platelet‐derived GF‐BB, which are well‐known regulators of tendon development, growth and healing. Among the screened hASCs subpopulations, tenomodulin‐positive cells were shown to be more promising for tenogenic applications and therefore this subpopulation was further studied, assessing tendon‐related markers (scleraxis, tenomodulin, tenascin C and decorin) both at gene and protein level. Additionally, the ability for depositing collagen type I and III forming extracellular matrix structures were weekly assessed up to 28 days. The results obtained indicated that tenomodulin‐positive cells exhibit phenotypical features of tendon progenitor cells and can be biochemically induced towards tenogenic lineage, demonstrating that this subset of hASCs can provide a reliable source of progenitor cells for therapies targeting tendon regeneration.