Bone Matrix Gelatin Research Articles

Effects of T-2 toxin and selenium on chondrocyte expression of matrix metalloproteinases (MMP-1, MMP-13), α2-macroglobulin (α2M) and TIMPs

T-2 toxin is regarded as an important etiological factor of Kashin-Beck disease, and supplementation of selenium–salt partly prevents Kashin-Beck disease. The present study investigated the effects of T-2 toxin on the degradation of type II collagen in human chondrocytes in vitro. Human chondrocytes were isolated and cultured on bone matrix gelatin to form an artificial cartilage model in vitro with or without T-2 toxin and selenium. Immunohistochemistry analyses showed that T-2 toxin decreased type II collagen staining and selenium appeared to prevent the decrease in type II collagen induced by T-2 toxin in engineered cartilage. Then, Western blot and RT-PCR analyses showed that an increase in MMP-13 and MMP-1 expressions, and a decrease in the expression of the general endoproteinase inhibitor (α2M) were induced by T-2 toxin. Gelatin reverse zymography showed that TIMP-1 and TIMP-2 levels were decreased in a dose-dependent manner after exposure of T-2 toxin. Selenium had a protective role by increasing the level of type II collagen protein through down-regulation of MMP-13 protein and mRNA expression and up-regulation of TIMP-1 and TIMP-2 expressions. These data suggest T-2 toxin induces cartilage matrix degradation by the up-regulation of MMP-13 and TIMP-1, and down-regulation of TIMP-2 and α2M expressions.

In vitro cartilage tissue engineering using cancellous bone matrix gelatin as a biodegradable scaffold

In this study, we constructed tissue-engineered cartilage using allogeneic cancellous bone matrix gelatin (BMG) as a scaffold. Allogeneic BMG was prepared by sequential defatting, demineralization and denaturation. Isolated rabbit chondrocytes were seeded onto allogeneic cancellous BMG, and cell–BMG constructs were harvested after 1, 3 and 6 weeks for evaluation by hematoxylin and eosin staining for overall morphology, toluidine blue for extracellular matrix (ECM) proteoglycans, immunohistochemical staining for collagen type II and a transmission electron microscope for examining cellular microstructure on BMG. The prepared BMG was highly porous with mechanical strength adjustable by duration of demineralization and was easily trimmed for tissue repair. Cancellous BMG showed favorable porosity for cell habitation and metabolism material exchange with larger pore sizes (100–500 µm) than in cortical BMG (5–15 µm), allowing cell penetration. Cancellous BMG also showed good biocompatibility, which supported chondrocyte proliferation and sustained their differentiated phenotype in culture for up to 6 weeks. Rich and evenly distributed cartilage ECM proteoglycans and collagen type II were observed around chondrocytes on the surface and inside the pores throughout the cancellous BMG. Considering the large supply of banked bone allografts and relatively convenient preparation, our study suggests that allogeneic cancellous BMG is a promising scaffold for cartilage tissue engineering.

Demineralized bone matrix gelatin as scaffold for tissue engineering

The aim of the present study was to do a comparative light microscopical and histochemical analysis of bone regeneration processes in rabbits, with the use of demineralized bone matrix gelatin scaffolds. Our histological results showed that no gelatin scaffold was extruded, indicating that neither body fluid nor blood swept the gelatin scaffold from the implantation site, nor was the gelatin scaffold biodegraded. Additionally, no abscess or inflammation of the peripheral osseous tissues at the implantation site was observed, suggesting that the implantation of the gelatin scaffold in the vertebra bone defect did not cause histopathology or exhibit malbiocompatibility with the peripheral osseous tissues.

Effects of moniliformin and selenium on human articular cartilage metabolism and their potential relationships to the pathogenesis of Kashin-Beck disease

To investigate the effects of mycotoxin moniliformin (MON) on the metabolism of aggrecan and type II collagen in human chondrocytes in vitro and the relationship between MON and Kashin-Beck disease (KBD). Human chondrocytes were isolated and cultured on bone matrix gelatin to form an artificial cartilage model in vitro with or without MON toxin. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of aggrecan and type II collagen in the cartilage was determined using immunocytochemical staining. MON toxin inhibited chondrocyte viability in dose-dependent and time-dependent manners. MON reduced aggrecan and type II collagen syntheses in the tissue-engineered cartilage. MON also increased the expression of matrix metalloproteinase-1 (MMP-1), MMP-13, BC4 epitopes, and CD44 in cartilages. However, the expression of 3B3(-) epitopes in cartilages was inhibited by MON. Selenium partially alleviated the damage of aggrecan induced by MON toxin. MON toxin promoted the catabolism of aggrecan and type II collagen in human chondrocytes.

Cartilage Tissue Engineering With Demineralized Bone Matrix Gelatin and Fibrin Glue Hybrid Scaffold: An In Vitro Study

To develop a cartilage-like tissue with hybrid scaffolds of demineralized bone matrix gelatin (BMG) and fibrin, rabbit chondrocytes were cultured on hybrid fibrin/BMG scaffolds in vitro. BMG scaffolds were carefully soaked in a chondrocyte-fibrin suspension, which was polymerized by submerging the constructs into thrombin-calcium chloride solution. Engineered cartilage-like tissue grown on the scaffolds was characterized by histology, immunolocalization, scanning electron microscopy, biochemical assays, and analysis of gene expression at different time points of the in vitro culture. The presence of proteoglycan in the fibrin/BMG hybrid constructs was confirmed by positive toluidine blue and alcian blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene-encoded cartilage-specific markers, collagen type II, and aggrecan core protein. The glycosaminoglycan production and hydroxyproline content of tissue grown on the fibrin/BMG hybrid scaffolds were higher than that of the BMG group. In conclusion, the fibrin/BMG hybrid scaffolds may serve as a potential cell delivery vehicle and a structural basis for cartilage tissue engineering.

A comparison of bone reconstruction following application of bone matrix gelatin and autogenous bone grafts to alveolar defects: An animal study

The aim of this study was to compare the quantity and quality of osseous healing of bone matrix gelatin (BMG) with autogenous bone graft (ABG) in cat models. Osseous defects (5 x 5mm) were made through upper alveolar bone distal to the canines in the left and right quadrants (maxilla) in each of twelve Persian male cats and filled randomly with BMG and ABG. The repair response was examined on days 14, 28, and 56 after surgery (n=4 per bone substitute per time point). Qualitative histological and quantitative histometric analysis including percentage of new formed bone fill (BF) and density were done. The inter-treatment comparison of mean levels of BF at specific time points showed consistently greater levels within BMG treated defects and reached significance on days 14, 28, and 56 (P<0.05). Regarding bone density (BD), there was no significant difference between BMG and ABG on days 14 and 28 (P>0.05). On day 56, BD was significantly higher within BMG group (P<0.05). Intra-treatment evaluation of bone formation in each group showed that BD significantly increased during treatment (P<0.05), and BF in BMG group reached significance at every time point but in ABG group only increase from day 28 to 56 reached significance (P<0. 05). Our results support the use of BMG to treat bone defects. Clinicians might expect better clinical outcomes without the possibility of side effects and morbidity associated with the harvest of autogenous bone.

Comparison of the Human Bone Matrix Gelatin (HBMG) with Autogenous Bone Graft in Reconstruction of the Parietal Bone Defects in Rat: A Histological and Radiographic Study.

Background and aims Autogenous bone graft is commonly used for reconstruction of bone defects in routine surgical procedures. The complexity of producing bone grafts and their application has lead to the use of human bone matrix gelatin (HBMG). The present study was conducted to compare the efficacy of HBMG and autograft on the reconstruction of bone defects in rats. Materials and methods In this cross-sectional, experimental study, two defects were put on left and right sides of parietal bone of rats. HBMG was placed randomly on defects of one side and autograft in the defects of the other side. All specimens were assessed and compared with each other according to histological and radiographic characteristics. Other assessments included amount and the rate of bone formation, inflammation signs, fibrosis tissue and cartilage formation and also radio-graphic characteristics of grafts, assessed by digital and film-based methods. Mann-Whitney U test was used for statistical analysis. Results The results showed a reduction of inflammation and an increase in new bone formation in both groups in 7, 14, 28 and 60 days after surgery. Bone formation with HBMG on day 24 was more than autograft. However, there was no sig-nificant difference between the groups on day 60. Superiority of digital method to film-based method of imaging was also observed. Conclusion Although HBMG has the same efficacy as autograft, the rate of bone reconstruction with HBMG is higher. HBMG also induces focal, rather than peripheral, bone construction in the defect.

Biphasic scaffold for annulus fibrosus tissue regeneration

Intervertebral disc (IVD) degeneration is the major cause of lower back pain, while the currently available treatments are symptomatic rather than curative. Tissue engineering is a powerful therapeutic strategy that can restore the normal biomechanical motion of the human spine. The ability of a biphasic elastic scaffold to structurally and elastically simulate the annulus fibrosus (AF) tissue of the IVD was explored. The outer phase of the scaffold was a ring-shaped demineralized bone matrix gelatin (BMG) extracted from cortical bone, which mimicks the type I collagen structure and ligamentous properties of outer AF. The inner phase of the scaffold was a bio-biomaterial poly(polycaprolactone triol malate) (PPCLM) orientated in concentric sheets and seeded with chondrocytes to recapitulate the inner layer of the AF, which is rich in type II collagen and proteoglycan. The mechanical properties and degradation of PPCLM could be adjusted by controlling the post-polymerization time of the pre-polymer. PPCLM also demonstrated good biocompatibility in a foreign body response in vivo assay. Incorporation of BMG into the scaffold enhanced the compressive strength compared with PPCLM alone. In addition, the tensile stress of the BMG/PPCLM scaffold was 50-fold greater than that of PPCLM alone, and close to that of normal rabbit AF. Finally, the biphasic scaffold supported the growth of rabbit chondrocytes, as confirmed by Safranin-O and type II collagen immunostaining. The excellent mechanical properties and biocompatibility of the BMG/PPCLM scaffold make it a promising candidate for AF repair.

Demineralized bone matrix gelatin as scaffold for osteochondral tissue engineering

To develop a single-unit osteochondral tissue with demineralized bone matrix gelatin (BMG), rabbit chondrocytes were cultured on demineralized bone matrix gelatin for 6 weeks. The engineered osteochondral tissue was characterized with histology, immunolocalization, TEM, SEM, biochemical assay, and gene expression analysis. About 1.3 mm viable neo-cartilage was produced on demineralized BMG. RT-PCR, immunohistochemistry, TEM, biochemical assay, and histology revealed hyaline-like cartilage with zonal layers, intense type II collagen expression, and abundant proteoglycan content formed upon BMG compared with normal cartilage. But hydroxyproline content and type I collagen gene and protein expressions were significantly lower. We consider engineering cartilage tissue with chondrocytes cultured on allogenic demineralized BMG is a good approach for osteochondral tissue engineering.

Repair of defects in articular joints

Repair of defects in articular joints

A study on antigen of heterogeneous bone matrix gelatin

目的 探讨异种骨基质明胶(BMG)的抗原性.方法临床植入异种BMG167例.对异种BMG抗原性行组织切片观察、扫描电镜观测及免疫组化检测;20例检测术前、术后1周、术后2周血清中与免疫应答相关的细胞因子,包括白介素2、4、6、10、12、转化生长因子-β1、肿瘤坏死因子-α.结果所有病例术后体温均在38.5℃以下,除1例外伤口均甲级愈合.组织切片示BMG成骨与正常骨结构一致;扫描电镜示BMG哈佛氏管和骨陷窝内未见血管内皮组织和骨细胞;免疫组化染色示BMG内没有可检出的抗原.细胞因子术前与术后比较,差异无显著意义(P＞0.05).结论异种BMG的抗原性极弱。

Demineralized bone matrix and native bone morphogenetic protein in orthopaedic surgery.

The recognition that demineralized bone matrix could induce bone formation when placed in mammalian skeletal muscle led to preclinical studies of crude native insoluble bone morphogenetic protein and noncollagenous protein, followed by the clinical application of demineralized bone matrix, chemosterilized autolyzed antigen-extracted allogenic bone, and autolyzed antigen-extracted allogenic bone matrix gelatin. Cultural norms and regulatory agencies influence the availability of different demineralized bone matrix preparations in different parts of the world, but there is continued interest in the biologic structure of native insoluble bone morphogenetic protein and noncollagenous protein aggregates and the applied science of osteoinduction and osteoconduction in reconstructive orthopaedic surgery. Demineralized bone matrix is not widely available in Asia, but tissue processing facilities in the United States distribute demineralized bone matrix materials with different carriers, handling properties, and possibly osteoinductive potential. The purpose of the current study was to review the development and use of various preparations of demineralized bone matrix materials.

Bone bonding in bioactive glass ceramics combined with bone matrix gelatin.

To determine how to encourage inductive osteogenesis on bioactive ceramics and accelerate the bonding of implants to the surrounding bone, we studied the role of autolyzed antigen-extracted allogeneic bone gelatin (AAAG) in bone bonding to bioactive ceramic implants in rabbit tibiae. Smooth-surfaced plates (15 x 10 x 2 mm) of apatite and wollastonite containing glass-ceramic were implanted into the proximal metaphyses of tibiae, with AAAG packed into the medullary cavity in one limb but not in the contralateral limb, which served as a paired control. After 2, 4, 8, 16, and 25 weeks, bone bonding and bone formation at the bone/implant interfaces were evaluated by a detaching test and undecalcified histological examination. The tensile failure load increased from 2 to 25 weeks for both groups. The failure load of the AAAG-treated group was significantly greater than that of the control group at every stage. Histologically, the AAAG-treated specimens showed active new bone formation in the medullary cavity and extensive bonding between the implant and bone at early periods. The percentage of bony covering in the AAAG-treated group was significantly higher than that of the controls at all intervals except at 25 weeks. The results of this study suggest that the addition of osteoinductive AAAG to a bioactive implant may significantly accelerate bone apposition to the implant and improve the bonding process at the interface, which would help to establish an earlier and stronger bonding between the implants and the surrounding bone.

Microassay for the assessment of low levels of hydroxyproline.

Microassay for the assessment of low levels of hydroxyproline.

A mushroom-shaped osteochondral patella allograft.

Replacement of the damaged chondral surface of the patella is often difficult because of the high mechanical stresses and the relatively thin cancellous surfaces. A new method has been developed to resurface the whole gliding surface with a mushroom-shaped allograft which provides firm fixation and an excellent base for rapid revascularisation. Fibrin gives additional stability and bonematrix gelatin (BMG) is used to enhance creeping substitution. Immediate motion and weightbearing promote rapid rehabilitation.

Effect of 1-hydroxyethane-1,1-diphosphonic acid (HEDP) on new bone formation induced by bone matrix gelatin (BMG)

The effect of the biphosphonate 1-hdroxyethane-1,1 diphosphonic acid (HEDP) on induced ectopic osteoneogenesis was studied by light and electron microscopy. Demineralized bone matrix gelatin (BMG) was implanted into the rectus abdominis muscle of 8-week-old Sprague-Dawley rats, HEDP was administered subcutaneously with a mini-osmotic pump, and histological changes were examined 4 days, 1, 2 and 3 weeks later. Four days after implantation, "acellular mineral deposition" in the control group was suppressed in the HEDP group. Two weeks after BMG implantation, cartilage formation was more intensive in the HEDP-treated group than in the control group. Three weeks after implantation, new lamellar bone with bone marrow was observed in the control group, while immature osteoid tissue detected by type II collagen in the central area was prominent in the HEDP-treated group. HEDP-treatment also suppressed the appearance and activity of osteoclast-like cell.

ＢＭＰ活性に及ぼすリン酸カルシウム系材料の影響について

In this experiment, five different soluble calcium phosphates ceramics were synthesized: HAP, β-TCP, α-TCP, OCP (octacalcium phosphate) and DCPD (dicalcium phosphate dehydrate). The effects of the ceramics on BMP activity were investigated in vitro and in vitro.Five mg of BMP was prepared along with 5 or 25 mg of ceramics. These were then combined to form complexes. These complexes were implanted epifascially in the femoral region, and their osteoinductive activities were examined at 4 weeks after implantation by means of roentogenographic and histologic observations. The amount of bone induced by the complexes was determined by a computer-supported image analysis system. For in vitro studies, a culture system which differentiated neonatal muscle into cartilage in response to BMP contained in Bone Matrix Gelatin (BMG (+)) was adopted. Calcium phosphate was applied onto the BMG, and a fetal rat muscle was placed on top, and the system was cultured. On Day 14 of culture, incorporation of [35S]-sulfate into glycosaminoglycans (GAG) was measured. Furthermore, to study the solubility of the ceramics in the culture medium, the ceramics were soaked in the same medium for 3 days, and the amounts of Ca and P and the change in pH were measured.HAP, β-TCP and α-TCP did not affect BMP activity when 5 mg and 25 mg ceramics/BMP complexes were implanted in the mice. OCP and DCPD did not affect BMP activity when 5 mg ceramics/BMP complexes were implanted, but inhibited BMP activity when 25 mg ceramics/BMP complexes were implanted. Only DCPD inhibited BMP activity while other ceramics did not in vitro. HAP, β-TCP and α-TCP produced no change in the Ca and P concentrations of the medium, but DCPD increased the concentrations of Ca and P and OCP decreased the concentrations of Ca and P. All the ceramics behaved similarly to the control in regard to the pH of the medium.It was concluded that HAP, β-TCP and α-TCP, which did not affect Ca and P concentrations in the medium, did not influence BMP activity. DCPD, which showed high solubility, inhibited BMP activity. Further investigations are necessary to determine the effect of apparent volume of OCP on BMP activity, and the effect of the phase transformation of OCP on solubility.

Early Osteoinduction in Rats Is Not Altered by Fibrin Sealant

The angiogenic and fibrogenic activities of fibrin sealant (Tisseel) suggest that it could enhance osteoinductivity during the first days after implantation. To test this hypothesis, the osteoinductivity of sealed and unsealed bone matrix gelatin was tested by implantation into rat abdominal wall muscle pouches for two to ten days. A sponge of collagen with or without fibrin sealant was used for control. Alkaline phosphatase (ALPase) was employed to quantify osteoinduction, and myeloperoxidase (MPO) was used to determine the inflammatory reaction. Plain histologic analysis of bone matrix gelatin implants at Days 2, 4, and 10 did not demonstrate any significant differences between sealed and nonsealed implants. Histologic analysis at Day 21 confirmed the osteoinductivity of the implant. The combination of bone matrix gelatin and fibrin sealant induced an early significant increase in ALPase activity, which could be interpreted as early induction of histologically undetectable osteogenesis. A simultaneous increase in the level of MPO suggested that the bone matrix gelatin/fibrin sealant-induced ALPase reaction might be caused by an inflammatory process, but neutrophil counting did not correlate to the MPO data. Morphometry, however, seems to be insufficient to assay a heterogenous histologic distribution of singular cells. It is proposed that the ALP/MPO ratio may be used as an inflammation-independent marker of osteogenesis. This study does not support the notion of an osteoinductive potency of fibrin sealant per se; however, it is clear that fibrin sealant does not impair bone matrix gelatin-dependent osteoinduction.

Experimental and Clinical Investigations of Human Insoluble Bone Matrix Gelatin A Report of 24 Cases

Human bone matrix gelatin (hBMG) was implanted into the quadriceps muscles of mice to determine its osteoinductive activity and immune responses. The host mouse muscle assay reacted positively to the hBMG, providing an experimental basis for clinical application. At two weeks after implantation, cartilage was produced in the mouse muscle, and new bone and bone marrow were formed at three to four weeks after implantation. Human bone matrix gelatin, which was thus shown to have osteoinductive activity, was subsequently implanted in 24 patients. Generally, the bone defects were filled with new bone tissue with increased density within two to four months after operation. Nonunion and delayed unions were healed at two to six months after surgery.

Ultrastructural observation of calcification preceding new bone formation induced by demineralized bone matrix gelatin.

Demineralized bone matrix gelatin (BMG) was implanted into the skeletal muscle of Sprague-Dawley rats, and the resulting ultrastructural changes of the BMG were examined 3, 5, 7, 10, 15 and 20 days later. Most of the implanted BMG became calcified 7-20 days after implantation. Calcification ('acellular mineral deposition') was first observed as needle-shaped crystalline deposits in the BMG matrix on day 7 after implantation, which gradually increased in size and fused with one another in some deposits. They appeared to be divided into small partitions consisting of denatured collagen fibers unlike those of noncalcified BMG. Some deposits had electron-lucent areas in their center adjacent to well-calcified peripheral areas, and the central area contained many collagen-like fibers and spherical vacuoles. Osteoblast-like cells were not present around these calcified deposits 7 days after implantation. After that, new bone formation was often seen near the area of acellular mineral deposition, and the fused calcified deposits remained until day 15 after implantation. The noncalcified BMG was mainly absorbed by macrophages, and the sites of acellular mineral deposition were absorbed by multinucleated cells resembling osteoclasts which were considered to be activated by the implants. Acellular mineral deposition probably started as calcium and phosphate deposits on some materials in the BMG matrix, and by heterogenic nucleation without osteoblasts or matrix vesicles, inducing bone formation. Thus the BMG may be not only a carrier of bone morphogenetic protein, gradually supplying it to the surrounding tissue but also a storage site for minerals that are indispensable for bone formation.(ABSTRACT TRUNCATED AT 250 WORDS)