Biodegradation of different synthetic hydrogels made of polyethylene glycol hydrogel/RGD-peptide modifications: an immunohistochemical study in rats

Aim: The aim of the present study was to investigate the pattern of biodegradation of different polyethylene glycol (PEG) hydrogel/RGD-peptide modifications in rats.
Material and methods: Two different hydrogels were employed: (i) a combination of four-arm PEG-thiol, M _n=2.3 kDa, and eight-arm PEG-acrylate, M _n=2.3 kDa (PEG1); and (ii) a combination of four-arm PEG-thiol, M _n=2.3 kDa, and four-arm PEG-acrylate, M _n=15 kDa (PEG2). Both PEG1 and PEG2 were either used alone or combined with a nine amino acid cys-RGD peptide (RGD). A non-cross-linked porcine type I and III collagen membrane [BioGide^® (BG)] served as control. Specimens were randomly allocated in unconnected subcutaneous pouches separated surgically on the back of 60 wistar rats, which were divided into six groups (1, 2, 4, 8, 16, and 24 weeks). Specimens were prepared for histological (tissue integration, foreign body reactions, biodegradation) and immunohistochemical (angiogenesis) analysis.
Results: All materials investigated revealed unimpeded and comparable tissue integration without any signs of foreign body reactions. While BG exhibited transmembraneous blood vessel formation at 1 week, all PEG specimens were just surrounded by a well-vascularized connective tissue. The hydrolytic disruption of PEG1 and PEG1/RGD specimens was associated with an ingrowth of blood vessels at 4 weeks. Biodegradation times were highest for PEG1 (24 weeks)>PEG1/RGD (16 weeks)>BG (4 weeks)>PEG2=PEG2/RGD (2 weeks).
Conclusion: Within the limits of the present study, it was concluded that (i) all materials investigated revealed a high biocompatibility and tissue integration, and (ii) hydrogel biodegradation was dependent on PEG composition.     

The behavior of human ameloblastoma tissue in collagen matrix in vitro: an immunohistochemical study

We investigated the three-dimensional outgrowth of human ameloblastoma cells cultured in collagen matrix. Growth patterns of four cases of ameloblastoma resembled each other and was characterized by appeared duct-like structures. Case 1 ameloblastoma was subcultured 3 times. Ameloblastoma tissues and cultured cells were stained with various lectins by avidin-biotin peroxidase complex (ABC) staining methods. Both the tissues and cultured cells had the same receptors to Concanavalin ensiforme (Con A), Ricinus communis Agglutinin (RCA-I) and Triticum vulgaris (WGA) but not to Dolichos biflorus (DBA), Ulex europaeus (UEA-1), Soybean (SBA) and Arachis hypogea (PNA). These findings indicated that the cultured cells examined in this study were ameloblastoma cells in origin.     

Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat

The aim of the present study was to compare the biodegradation of differently cross-linked collagen membranes in rats. Five commercially available and three experimental membranes (VN) were included: (1) BioGide (BG) (non-cross-linked porcine type I and III collagens), (2) BioMend (BM), (3) BioMendExtend (BME) (glutaraldehyde cross-linked bovine type I collagen), (4) Ossix (OS) (enzymatic-cross-linked bovine type I collagen), (5) TutoDent (TD) (non-cross-linked bovine type I collagen, and (6-8) VN(1-3) (chemical cross-linked porcine type I and III collagens). Specimens were randomly allocated in unconnected subcutaneous pouches separated surgically on the back of 40 wistar rats, which were divided into five groups (2, 4, 8, 16, and 24 weeks), including eight animals each. After 2, 4, 8, 16, and 24 weeks of healing, the rats were sacrificed and explanted specimens were prepared for histologic and histometric analysis. The following parameters were evaluated: biodegradation over time, vascularization, tissue integration, and foreign body reaction. Highest vascularization and tissue integration was noted for BG followed by BM, BME, and VN(1); TD, VN(2), and VN(3) showed prolongated, while OS exhibited no vascularization. Subsequently, biodegradation of BG, BM, BME and VN(1) was faster than TD, VN(2), and VN(3). OS showed only a minute amount of superficial biodegradation 24 weeks following implantation. Biodegradation of TD, BM, BME, VN(2), and VN(3) was associated with the presence of inflammatory cells. Within the limits of the present study, it was concluded that cross-linking of bovine and porcine-derived collagen types I and III was associated with (i) prolonged biodegradation, (ii) decreased tissue integration and vascularization, and (iii) in case of TD, BM, BME, VN(2), and VN(3) foreign body reactions.     

Angiogenesis pattern of native and cross-linked collagen membranes: an immunohistochemical study in the rat

The aim of the present study was to immunohistochemically evaluate angiogenesis pattern of native and cross-linked collagen membranes after subcutaneous implantation in rats. Five commercially available and three experimental membranes (VN) were included: (1) BioGide (BG), (2) BioMend (BM), (3) BioMend Extend (BME), (4) Ossix (OS), (5) TutoDent (TD), and (6-8) VN(1-3). Specimens were randomly allocated in unconnected subcutaneous pouches (n=4) separated surgically on the back of 40 wistar rats, which were divided into five groups (2, 4, 8, 16, and 24 weeks), including eight animals each. Pattern of angiogenesis was labelled using primary mouse monoclonal antibody to transglutaminase II. For each membrane, the period of time, needed for a complete and homogeneous transmembraneous vascularization, was assessed immunohistomorhometrically. Differences between the membranes were found regarding the initial pattern of transmembraneous angiogenesis, as evaluated 2 weeks following implantation. Mean cross- and longitudinal-sectional area of blood vessels (%) was highest for VN(3) (5.27+/-2.73), followed by BG (2.45+/-0.88), VN(1) (2.07+/-0.29), VN(2) (1.91+/-0.55), TD (1.44+/-0.53), BME (0.35+/-0.29) and BM (0.25+/-0.4). In contrast to BG and VN(1-3), BM, BME and TD exhibited a homogeneous transmembraneous formation of blood vessels merely 4-8 weeks following implantation. OS, however, exhibited no signs of angiogenesis throughout the whole study period. Within the limits of the present study, it may be concluded that pattern of transmembraneous angiogenesis markedly differs among the membranes investigated.     

Enamel matrix derivative exhibits angiogenic effect in vitro and in a murine model

OBJECTIVES: Angiogenesis is one of the most critical events in the wound healing process. Any increase in angiogenesis could result in more rapid and complete healing. A recent study found that enamel matrix derivative (EMD) could accelerate early periodontal wound healing. We wanted to clarify whether EMD caused an angiogenic effect and, thus, possibly enhanced wound healing. METHODS: We performed in vitro proliferation and chemotaxis assays on human umbilical vein endothelial cell (HUVEC) cultures, and a tissue culture assay using blood vessel fragments in fibrin gel. Collagen membranes soaked with EMD were implanted subcutaneously in mice to test the in vivo angiogenic effect. RESULTS: While there were no significant differences between the negative control and EMD groups in the proliferation assay, EMD treatment did exhibit a significantly greater dose-dependent chemotactic effect on HUVEC than control group treatments. The tissue culture in fibrin gel showed new blood vessel outgrowths in the EMD groups, but none in the negative control group. In the animal studies, significantly more endothelial cells were detected in the EMD group of mice. CONCLUSIONS: Our findings show that EMD does exhibit some angiogenic effects. However, the underlying molecules and mechanisms are still unidentified. We discuss several possibilities.     

Behaviour of human ameloblastoma cells in collagen matrix in vitro: an ultrastructural study

We investigated the behaviour of human ameloblastoma cells in collagen matrix in vitro. The ameloblastoma tissue was extirpated from a 75 yr-old woman. Small segments of the tissues were cultured in 0.18% collagen gel. After several weeks, collagen gels containing ameloblastoma tissue were fixed and examined by light microscopy and transmission electron microscopy. Outgrowth of primary cultured cells could be recognized as processes like growth rims around the tissue pieces within 2-3 days after tissue culture. Light microscopically, these cells formed a duct-like structure. The characteristics of ameloblastoma cells in vivo with peripheral epithelial cells and stellate cells reappeared in vitro, and they were connected with one another in cytoplasmic processes by desmosomes. Also, some cells had many intracellular filaments, amorphous nuclei and vacuoles. The results indicated that this culture system in vitro might be applied to the cytogenetic analysis of ameloblastoma.     

牙本质内基质金属蛋白酶的测定及其对牙本质胶原降解作用的研究

[摘要] 目的 检测并研究冠根部的牙本质内基质金属蛋白酶(MMPs)及其对牙本质胶原纤维降解的作用。方法 将牙本质粉经盐酸胍提取,然后经EDTA循环脱矿,再经盐酸胍提取。提取物经免疫印迹与酶谱分析检测基质金属蛋白酶-2,9(MMP-2,9)及酶的活性。扫描电镜观察脱矿及脱矿后置于人工唾液中的冠根部牙本质表面结构变化。结果 免疫印迹结果显示提取物中含有MMP-2,9。酶谱分析结果显示提取物中MMP-2,9均具有活性。扫描电镜结果表明,脱矿的冠根部表面胶原纤维较完整;而脱矿后置于人工唾液中的牙本质表而胶原纤维断裂,结构紊乱。结论 冠根部牙本质中含有活性的MMP-2,9。脱矿过程中的低pH值能使牙本质中的MMP活化,在中性时可降解胶原纤维。     

Histopathological changes in collagen and matrix metalloproteinase levels in articular condyle of experimental model rats with jaw deformity

OBJECTIVE: To investigate the dynamics of the cartilage matrix in the articular condyle after removal of a side shift plate; Emergence of type I, II, and III collagen in the matrix as well as changes in levels of matrix metalloproteinase (MMP)-1, -8, and -13 that degrade collagen were studied histopathologically and immunohistochemically. DESIGN: Lateral displacement of the mandible was achieved by attaching a side shift plate to the anterior teeth of the maxilla in male rats at 6 weeks. The wearing period of the side shift plate was 8 weeks. Observations were made at 0, 1, 2, 4 and 8 weeks after removal. RESULTS: In histopathological findings, the timing of proliferation of the layer of hypertrophy varied between the bilateral sides. In immunohistochemical findings a significant decline in the expression of type II collagen in the displacement side was observed immediately after removal. Moreover, the expressions of MMPs were elevated in both sides on 0 weeks. At 1 week after removal, a significant elevated in the expression of type II collagen, MMPs was decline in both sides. CONCLUSIONS: After removal, the levels of MMP-1, -8, and 13 were reduced and the emergence of type II collagen increased. Thus, cellular outgrowth was initiated to trigger intracartilaginous ossification to restore the cartilage matrix.     

Healing of onlay mandibular bone grafts covered with collagen membrane or bovine bone substitutes: a microscopical and immunohistochemical study in the sheep

The objective of this study was to evaluate the role of collagen membrane and Bio-Oss coverage in healing of an onlay graft to the mandible. Twelve adult sheep each received an onlay bone graft (experiment 1), bone graft+Bio-Gide (experiment 2), and bone graft+Bio-Oss/Bio-Gide (experiment 3) on the lateral surface of the mandible. The animals were euthanized at 4, 8, 12 or 16 weeks after surgery, and findings were analysed by routine microscopy and immunohistochemistry for proliferation (Ki67) and apoptotic (Caspase-3) markers. Grafts were fully incorporated in all specimens. Pronounced resorption was observed in experiment 1. Minimal loss of graft volume was seen in experiment 2 specimens without membrane displacement. A remarkable increase in the augmented region of the mandible was observed in experiment 3. A high number of osteoclasts were expressed within the grafts during the early healing period, and thereafter declined markedly. Osteoblasts within the grafts expressed a moderate level of Ki67 at 8 weeks, which thereafter declined markedly. The strongest expression of Caspase-3 on the bone surface was observed after 16 weeks. In conclusion, the effect of collagen membrane coverage on bone graft volume maintenance was dependent on membrane stability during healing. An autogenous bone graft covered with Bio-Oss particles resulted in a remarkable increase in augmented lateral surface of the mandible. The late stage of bone graft healing was associated with a high apoptotic induction pathway of osteoblasts lining the surfaces of the new bone, demonstrated by strong positive Caspase-3 immunoreactivity.     

Clinical efficacy of xenogeneic collagen matrix in the treatment of gingival recession: a systematic review and meta-analysis

Objective: The aim of this systematic review (SR) was to evaluate the effects of xenogenic collagen matrix (XCM) on the outcomes of clinical treatments of patients with Miller class-I or -II gingival recessions.

Materials and methods: Articles that were published before March 2018 were electronically searched in four databases without any date or language restrictions and manually searched in regular journals and gray literature. The eligibility criteria comprised randomized controlled trials (RCTs) and prospective controlled trials with follow-up periods of 6 months or more that compared the performance of XCM in the treatment of Miller class-I or -II gingival recessions. This SR was registered in PROSPERO under number CRD42018106118.

Results: Nine RCTs published between 2010 and 2018 were included in this SR. The percentage of root coverage (RC) was significantly higher (p?=?.0003) when gingival recessions were treated with XCM when compared to coronally advanced flap (CAF) alone. In addition, the parameters of keratinized mucosa width (KMW) (p?=?.006) and gingival thickness (GT) (p?=?.0003) were also improved when the XCM was used in comparison to the CAF alone. There was not a statistically significant difference (p?=?.22) between the clinical attachment level (CAL) achieved with the use of XCM and that achieved with CAF alone. RC with the use of XCM, when compared to connective tissue grafts (CTGs) (p?=?.09) and enamel matrix derivative (EMD) (p?=?.62), there was no significant difference; however, XCM yielded lower RC than CTG in the treatment of Miller class-I or -II gingival recessions.

Conclusions: Based on both the individual studies’ outcomes and the pooled estimates, it can be concluded that the use of XCM improves the RC, KMW and GT in the treatment of gingival recessions when compared to CAF alone and may be a viable alternative to use of CTG.     

An immunohistochemical study of matrix molecules associated with barrier membrane-mediated periodontal wound healing

Guided tissue regeneration (GTR) is a clinical procedure developed to facilitate periodontal regeneration by using barrier membranes to selectively promote the repopulation of a periodontal defect by periodontal ligament and bone cells at the expense of epithelial and gingival connective tissue cells. The aim of this study was to gain insight into the biological events occurring during membrane mediated periodontal wound healing by examining the immunohistochemical expression of a number of extracellular matrix components in tissues treated via the GTR technique. Experimental periodontal defects were created around the second premolar tooth in 4 dogs and wound closure was achieved by application of expanded polytetrafluoroethylene membranes around each tooth and flap positioning coronal to the cementoenamel junction. The dogs were sacrificed after a 4-wk healing period, block dissections of the part of the mandible containing the experimental tooth were obtained and paraffin sections were prepared. Using standard immunohistochemical techniques, the sections were stained with a monoclonal antibody against bone morphogenetic proteins 2 and 4 (BMP-2 and -4) and polyclonal antibodies against collagen I, collagen II, decorin, biglycan, bone sialoprotein, osteopontin and osteocalcin. Collagen I was predominantly localized within the regenerating bone, whereas collagen III staining was more abundant in the soft connective tissues of the defect. Decorin and biglycan staining was faint within the extracellular matrix of the regenerating defect, although both proteoglycans exhibited intense intracellular localization within some of the cells inhabiting the defect. The staining for BMP-2 and -4 was weak within the bone but strong within the extracellular matrix of the regenerating soft tissue. Osteopontin and bone sialoprotein were strongly localized in the regenerating bone and cementum found within the defect. Osteocalcin staining was present in both the regenerating and mature cementum and associated cementoblasts, and it was relatively weaker in the regenerating bone compared to the mature bone. The observed pattern of immunolocalization of the extracellular matrix macromolecules suggests that the heterogeneous cell population filling the GTR wound had created an environment that was conducive to periodontal regeneration.     

Effect of platelet-released growth factors and collagen type I on osseous regeneration of mandibular defects. A pilot study in minipigs

OBJECTIVE: To study the effects of platelet-released growth factors (PRGF) and collagen type I on bone defect healing in minipig mandibles. MATERIAL AND METHODS: In eight adult minipigs defects were trephined in the facial mandibular wall from extra-oral and filled with collagen+PRGF or with collagen alone. Control defects were left untreated. PRGF were defined as the supernatants obtained after centrifugation of washed, thrombin-activated allogenic cells of platelet-rich plasma. The animals were sacrificed at 4 and 8 weeks. For histological analysis, undecalcified ground specimens stained with the Levai-Laczko stain were used. RESULTS: For the entire follow-up, the amount of newly formed bone was 35.49 +/- 3.84% in the collagen+PRGF group, 46.34 +/- 3.84% in the collagen-only group and 33.83 +/- 4.11% in the controls. The differences between the collagen+PRGF and the collagen-only group (p = 0.0343), and between the collagen-only group and the controls (p = 0.0305) were significant. Histologically, defects filled with collagen+PRGF showed inflammatory reactions at 4 weeks, and new bone formation near the remnants of the filler collagen was reduced. CONCLUSION: The data suggest that collagen type I alone, but not its combination with PRGF can support the early stages of cortical bone repair.