Tamoxifen对真皮成纤维细胞种植胶原网格的抑制效应

目的 探索Tamoxifen用于治疗皮肤瘢痕挛缩的可能性。方法 将人真皮成纤维细胞种植于Ⅰ型胶原构成的胶原网格内培养，加入1－50μmol/L的Tamoxifen，测定网格收缩率；采用MTT染色法原位观察网格内的细胞形态及活力变化。结果 Tamoxifen对胶原网格收缩具有抑制效应，其浓度低于5μmol/L时对网格收缩无影响，高于30μmol/L时网格收缩被不可逆性完全抑制，处于10－20μmol/L时对网格收缩作用呈现剂量及时间依赖性抑制。MTT染色后观察显示网格收缩变化与处于三维培养的细胞突起伸展状态相关；Tamoxifen并不直接导致网格内的细胞死亡，而是通过抑制细胞功能发挥作用。结论 Tamoxifen可对真皮成纤维细胞体外模拟的瘢痕挛缩产生抑制效应，提示对活体瘢痕挛缩具有潜在的抑制作用。     

Elucidating the mechanism of wound contraction: Rapid versus sustained myosin ATPase activity in attached-delayed-released compared with free-floating fibroblast-populated collagen lattices

Wound contraction closes open wounds by the generation of contractile forces within granulation tissue. We investigated the mechanism of wound contraction using the in vitro fibroblast-populated collagen lattice (FPCL) contraction model. The contraction of the free-floating (FF)-FPCL is through rapid myosin ATPase activity, while the contraction of the attached-delayed-released (ADR)-FPCL is through sustained myosin ATPase activity. All FPCLs were cast identically and the contraction of FF-FPCLs was recorded daily for 4 days and the contraction of ADR-FPCLs was recorded 1 hour after release on day 4. At day, 4 cell numbers were determined and cells undergoing apoptosis were identified and counted. Differences in sustained and rapid myosin ATPase activity were shown by added inosine triphosphate-induced cell contraction in permeabilized fibroblast monolayer preparations. At 2 days, the FF-FPCLs were mostly contracted, while an ADR-FPCL completed contraction 1 hour after release at day 4. Contracted myofibroblasts, identified by alpha-smooth muscle actin-stained stress fibers, were identified in contracted ADR-FPCL, whereas elongated fibroblasts were identified in contracted FF-FPCLs. Vanadate inhibited both inosine triphosphate-induced cell contraction and ADR-FPCL contraction, but neither inhibited ATP-induced cell contraction or FF-FPCL contraction. Genistein inhibited FF-FPCL contraction, but not ADR-FPCL contraction. Advancing tyrosine phosphorylation in fibroblasts promotes rapid myosin ATPase activity, while advancing tyrosine dephosphorylation in myofibroblasts promotes sustained myosin ATPase. The ADR-FPCL had a reduced cell count and a greater proportion of cells had entered apoptosis compared with FF-FPCL. These experiments show that FF-FPCL contraction is through elongated fibroblasts and rapid myosin ATPase, requiring tyrosine phosphorylation. In contrast, the mechanism for ADR-FPCL contraction is through cell contraction by sustained myosin ATPase, involving tyrosine dephosphorylation.     

The fibroblast-populated collagen matrix as a model of wound healing: a review of the evidence.

The fibroblast-populated collagen matrix (FPCM) has been utilized as an in vitro model of wound healing for more than 2 decades. It offers a reasonable approximation of the healing wound during the phases of established granulation tissue and early scar. The gross and microscopic morphology of the FPCM and the healing wound are similar at analogous phases. The processes of proliferation, survival/apoptosis, protein synthesis, and contraction act in similar directions in these two models, and the response to exogenous agents also is consistent between them. If its limitations are respected, then the FPCM can be used as a model of the healing wound.     

A study to the fibroblast—populated collagen lattices

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p27基因转染对增生性瘢痕成纤维细胞-胶原网收缩的影响

目的：研究p27对增生性瘢痕成纤维细胞-胶原网收缩的影响差异，探讨p27在瘢痕形成中的作用。方法：取体外培养的人增生性瘢痕成纤维细胞（HTsFb），p27基因转染HTsFb，建立成纤维细胞-胶原网模型，观察p27基因转染对p27基因转染HTsFb-胶原网收缩的影响差异。结果：p27能抑制HTsFb-胶原网收缩。结论：p27可能通过抑制瘢痕收缩来抑制瘢痕形成。     

Modulation of FAK, Akt, and p53 by stress release of the fibroblast-populated collagen matrix

BACKGROUND: Fibroblast survival in a three-dimensional collagen matrix is dependent in part upon the rigid anchorage of the matrix to tissue culture plastic. We hypothesized that focal adhesion kinase (FAK) and protein kinase B (Akt) would be activated and that the p53 level would be low in the rigidly anchored (attached) collagen matrix; loss of anchorage (detachment) was hypothesized to have the opposite effects. MATERIALS AND METHODS: Human foreskin fibroblasts were cultured in attached bovine collagen matrices for 48 h before detachment as free-floating matrices. At various time points postrelease, matrix lysates were blotted for the proteins of interest, and the terminal deoxynucleotidyltransferase-mediated dUTP nick-end label assay was performed on both whole matrices and cytospin preparations. Irradiated monolayer fibroblasts were used as positive controls for the amount of p53 protein. RESULTS: Terminal deoxynucleotidyltransferase-mediated dUTP nick-end label positivity in attached versus detached matrices (at 24 h post detachment) was 0.7 +/- 03 versus 5.3 +/- 1.7% (P < 0.05, unpaired t test). FAK and Akt were phosphorylated (activated) in the attached matrix; there was a near complete of loss of both activated forms within 4 h of matrix detachment. Irradiated monolayer fibroblasts had increased levels of p53, mdm2, and p21. In contrast, the p53, mdm2, and p21 levels were just at the level of detection in the attached matrix, but were induced 5- to 10-fold within 2-4 h after matrix detachment. CONCLUSIONS: FAK and Akt are activated in the attached fibroblast-populated collagen matrix whereas the p53 level is relatively low; matrix detachment downregulates FAK and Akt activity and induces p53. The state of mechanical anchorage of the collagen matrix regulates the survival of embedded fibroblasts through a mechanism which may involve FAK.     

Renal myofibroblasts contract collagen I matrix lattices in vitro

Myofibroblasts, cells with both fibroblastic and smooth muscle cell features, have been implicated in renal scarring. In addition to synthetic properties, contractile features and integrin expression may allow myofibroblasts to rearrange and contract interstitial collagenous proteins. Myofibroblasts from normal rat kidneys were grown in cell-populated collagen lattices to measure cell generated contraction. Following detachment of cell populated collagen lattices, myofibroblasts progressively contracted collagen lattices, reducing lattice diameter by 42% at 24 h. Alignment of myofibroblasts, rearrangement of fibrils and beta(1) integrin expression were observed within lattices. We postulate that interstitial myofibroblasts contribute to renal scarring through manipulation of collagenous proteins. Copyright Copyright 1999 S. Karger AG, Basel     

Ultrastructural changes during contraction of collagen lattices by ocular fibroblasts

Contraction and scarring of the cornea and conjunctiva following disease or injury are major causes of visual morbidity. The aim of this study was to identify any specific ultrastructural features of ocular fibroblast behavior in different collagen lattices in order to understand some of the mechanisms of cell-mediated contraction. Normal human Tenon's capsule fibroblasts were cultured within both restrained and floating collagen lattices for periods of up to 13 days and then analyzed using transmission electron microscopy. The contractile force of these fibroblasts was also tested using the culture force monitor, an instrument capable of measuring the minute forces exerted by cells within a collagen lattice. The results showed differences in the behavior of fibroblasts cultured in the two gel models. The features seen in restrained gels suggest that fibroblasts were actively migrating across and through the lattice. These migratory features were not seen to the same extent in untethered gels, which lack the inherent tension and support of the tethered model. We hypothesize that contraction of the collagen matrix in tethered lattices is due to cellular migration and that this fact cannot be ascertained from untethered gels. Both lattice models have experimental value, but it is important to appreciate what mechanical signals cells receive from the matrix in order to understand cellular behavior.     

Mineralization and alkaline phosphatase activity in collagen lattices populated by human osteoblasts

Adult human osteoblastic cells were grown in a native type I collagen gel. Proliferation and viability analyses showed that cells rapidly stopped dividing and became blocked in the G0G1 phase (91% on day 13). Carboxyfluorescein diacetate cell staining and flow cytometry showed that osteoblasts were viable for the first 16 days and then viability decreased (58% viable cells on day 22). Osteoblasts were able to retract the matrix. Betaglycerophosphate (βGP) stimulated the deposition of mineral particles in the collagen network, and electron probe microanalysis showed that they were principally calcium and phosphorus, with a Ca/P ratio of about 1.7. Various times of βGP supply were tested. We compared 10 mM βGP added only once at day 0, or continuously from day 0, day 8, or day 21. Mineralization was observed in conditions where βGP was added at day 0. Furthermore, 10 mM βGP added once during gel preparation was sufficient to induce mineralization with mineral accumulation up to day 15 whereas the speed of the gel contraction decreased. In every condition, cultures expressed high alkaline phosphatase (ALP) levels as early as day 3, which decreased afterwards. These kinetics might explain why the other conditions did not prove favorable to the mineralization process. The model was used to study the influence of blocking gel retraction. Blocking retraction delayed the ALP activity decrease, but had no effect on mineralization. In conclusion, human adult osteoblasts cultured in native collagen gel stopped proliferation and underwent mineralization very early. This model should be used to investigate the influence of effectors on the early stages of culture. Received: 15 October 1997 / Accepted: 1 July 1999     

Human renal fibroblast contraction of collagen I lattices is an integrin-mediated process.

BACKGROUND: Expression of the beta1 family of integrins allows dermal fibroblasts in wounds to contribute to the healing process through migration, adhesion, synthesis, and rearrangement of extracellular matrix. To date the ability of human renal fibroblasts to reorganize collagens and the role of cell surface receptors in this process remain unknown. METHODS: Renal fibroblasts were grown from the cortical tissue of surgically removed human kidneys. The ability of human renal fibroblasts to reorganize interstitial collagen I was examined in vitro using solidified collagen I lattices. Integrin function was blocked by incubating fibroblasts with isotype-specific antibodies prior to addition to collagen I lattices. RESULTS: Human renal fibroblasts embedded in collagen I lattices progressively decreased lattice diameter to 60.6+/-11.4% of initial diameter at 48 h post-release (P:<0.01). Fibroblasts incubated in the presence of antibody to beta1 integrin failed to contract collagen I lattices, whilst fibroblasts incubated with non-specific antibody reduced lattice diameter to 60.1+/-12.4% of initial diameter at 48 h post-release (P:<0.01). Further characterization of integrin alpha subunits showed that blocking alpha2beta1 integrin prevented lattice contraction (P:<0.05, alpha2beta1 integrin antibody vs non-specific antibody), whilst blocking of alpha5beta1, alpha3beta1 and alpha1beta1 integrins did not influence this process. CONCLUSIONS: We postulate that collagen I fibril rearrangement by human renal fibroblasts in vitro appears to be an integrin-mediated process involving the alpha2beta1 integrin.     

Calmodulin-myosin light chain kinase inhibition changes fibroblast-populated collagen lattice contraction, cell migration, focal adhesion formation, and wound contraction

Wound healing requires fibroblast migration, synthesis of new extracellular matrix, and organization of that matrix, all of which depend upon myosin ATPase activation and subsequent cytoplasmic actin-myosin contraction. Myosin ATPase activity is optimized by phosphorylation of myosin light chain at serine 19. Several different signaling pathways can perform that phosphorylation, the focus here is calcium saturated calmodulin dependent -myosin light chain kinase (CaM-MLCK). It is proposed that CaM-MLCK phosphorylation of myosin light chain and subsequent myosin ATPase activation affects granulation tissue fibroblast behavior and contributes to wound contraction. Myosin ATPase activity generates actin-myosin contraction within fibroblasts. Myosin ATPase activity is involved in ATP-induced cell contraction, the generation of focal adhesions, fibroblast migration, fibroblast populated collagen lattice (FPCL) contraction, and wound contraction. The MLCK inhibitors ML-9 and ML-7 inhibited ATP-induced cell contraction, fibroblast migration, FA formation, and FPCL contraction. The calmodulin inhibitors W7 and fluphenazine blocked rat open wound contraction. In addition, fluphenazine delayed re-epithelialization. These findings support the idea that fibroblast CaM-MLCK activity is essential for tissue repair. We speculate that inhibition of CaM-MLCK may reduce or prevent detrimental fibrotic contracture.     

In vitro fibroblast populated collagen lattices are not good models of in vivo clinical wound healing

In chronic wounds, the healing process is prolonged and incomplete, proceeding in an uncoordinated manner, and resulting in poor anatomical and functional outcome. There have been numerous attempts to discover models that mimic human wound healing processes. The fibroblast populated collagen lattice is one such model that has been proposed. This study evaluated whether the fibroblast populated collagen lattice can be a model of chronic wound healing using the pressure ulcer as a paradigm. Fibroblast cultures of wound biopsies and wound volume measurements were obtained serially during a four arm blinded, placebo-controlled sequential cytokine clinical trial of pressure ulcers. Fibroblasts obtained from study patients were added to collagen lattices and contraction was determined daily for 10 days. Collagen gel-area measurements were converted to reflect percentage of gel contraction. These data of both edge and base wound biopsies on days 0, 10, and 36 were categorized into treatment groups and one-way analysis of variance showed no significant differences in contraction among these groups. When considering all fibroblast populated collagen lattices, there was significantly greater contraction at days 10 and 36 for cells from both edge and base biopsies compared to day 0 (p < 0.05). The Spearman Rank Correlation test comparing all patients with fibroblast populated collagen lattice results from fibroblasts obtained at the edge or base of the wound at days 0, 10, and 36 and clinical pressure ulcer healing on day 36 showed no correlation. This lack of correlation not only persisted for each of the four treatment arms but also for responder status based on decrease in wound volume over the 35 day trial period. In conclusion, chronic wound healing is a complex process that is not modeled by in vitro fibroblast populated collagen lattices.     

重组人核心蛋白多糖固相拮抗转化生长因子β1对瘢痕成纤维细胞的刺激作用

目的模拟人体内核心蛋白多糖和转化生长因子β1(TGF-β1)接触方式,观察核心蛋白多糖固相拮抗TGF-β1刺激瘢痕成纤维细胞的效果.方法制备成纤维细胞胶原网格(FPCL)体外三维培养模型,将其分为4组.对照组向FPCL中加入培养液;核心蛋白多糖组向FPCL中混入终浓度2 mg/L的重组人核心蛋白多糖,再加入培养液;TGF-β1组向FPCL中加入含5 μg/L TGF-β1的培养液;TGF-β1+核心蛋白多糖组向FPCL中混入终浓度2 mg/L的重组人核心蛋白多糖,然后加入含5μg/L TGF-β1的培养液.在培养12、24、48、72、96 h时观察各组FPCL的收缩情况,并用蛋白质印迹法与逆转录聚合酶链反应分别检测FPCL中瘢痕成纤维细胞Ⅰ型纤溶酶原激活物抑制因子1(PAI-1)、α平滑肌肌动蛋白(α-SMA)的蛋白及mRNA表达水平.结果各培养时相点下,TGF-β1组FPCL收缩比对照组明显增强,核心蛋白多糖组FPCL收缩则比对照组明显减弱.TGF-β1组的PAI-1、α-SMA的蛋白及相应mRNA表达水平(3 482±211、4 320±272;0.89±0.15、0.56±0.11)显著高于对照组(1 764±147、1 699±146;0.29±0.06、0.21±0.06,P＜0.01);其余两组相应检测指标与对照组比较差异无统计学意义(P＞0.05).结论重组人核心蛋白多糖混入胶原凝胶,可显著抑制TGF-β1对瘢痕成纤维细胞的刺激作用,表明在体外核心蛋白多糖具有拮抗TGF-β1的作用.提示皮肤组织损伤后,由于创面机械性缺少核心蛋白多糖,TGF-β1活性上调,可能是瘢痕增生的一个重要因素.     

A collagen coated vascular prosthesis

A woven, double velour Dacron vascular graft was made nonporous by coating it with a layer of collagen prepared from fresh, young calf skin. Grafts were implanted in the thoracic aorta of 24 mongrel dogs and were examined at intervals up to 180 days. The grafts did not require preclotting or special preparation before being implanted. They sutured easily and did not bleed. When explanted all grafts were patent and covered with neointima. The bovine collagen was almost completely resorbed by 90 days and was replaced with native tissue. The collagen was neither thrombogenic, antigenic, cytotoxic, or pyrogenic.     

A preliminary characterization of human cementum collagen

Summary Human teeth were used to obtain cementum. Collagen could not be significantly solubilized from the cementum by salt and acetic acid extraction or by pepsin digestion. CNBr digestion (86%) of cementum and subsequent carboxymethyl cellulose chromatography suggests that human cementum consists of type I collagen only as identified by amino acid and hexose analyses.     

Clinical outcomes and complications of a collagen meniscus implant: a systematic review

Purpose

The purpose of this systematic review was to summarise and evaluate the clinical outcomes of the collagen meniscus implant (CMI) and its complication and failure rates. These data were then used to evaluate the results of the CMI at different follow-up time periods and investigate possible differences in the behaviour of lateral and medial CMI.

Methods

A comprehensive search was performed in PubMed, MEDLINE, CINAHL, Cochrane, EMBASE and Google Scholar databases using various combinations of the following keywords: “collagen meniscus implant” or “collagen meniscal implant”. All studies evaluating medial or lateral CMI using the Lysholm score, visual analogue scale (VAS) for pain, Tegner activity scale and subjective or objective International Knee Documentation Committee (IKDC) scores were included in the systematic review.

Results

Eleven studies were included in the systematic review. The pooled number of patients involved in CMI surgery were 396 (90.2 % medial, 9.8 % lateral), with a mean age at surgery of 37.8 years. Concomitant procedures were present in 48.8 % of patients; most of them were anterior cruciate ligament (ACL) reconstruction, high tibial osteotomy (HTO) and microfractures. The Lysholm score and VAS for pain showed an improvement at six months up to ten years. No noticeable differences were present comparing short-term values of Lysholm score between medial and lateral CMI. The Tegner activity level reached its peak at 12 months after surgery and showed a progressive decrease through five and ten years post CMI implantation, however always remaining above the pre-operative level. Only a few knees were rated as “nearly abnormal” or “abnormal” at IKDC grading at all follow-up evaluations.

Conclusions

The CMI could produce good and stable clinical results, particularly regarding knee function and pain, with low rates of complications and reoperations.