Evaluation of demineralised,freeze-dried,irradiated bone allografts in the treatment of osseous defects in the oral cavity

Demineralised, freeze-dried bone allografts (DFDBA) have been used extensively by dentists in the treatment of periodontal and periapical osseous defects resulting from inflammatory diseases. Their use in India however, is limited by the availability of quality allografts and the high cost of imported alternatives. A study was conducted to assess the osteogenic potential of DFDBA prepared for the first time in India by the Tata Memorial Hospital (TMH) Tissue Bank. The DFDBA was used in the treatment of osseous defects after removal of periapical lesions associated with devitalised teeth in 10 healthy patients. At the 6-month recall visit all the patients showed a remarkable decrease in the grades of mobility, and 9 out of the 10 patients showed radiographic evidence of complete healing of the osseous defects with evidence of normal bony trabaeculae. These findings indicate that the indigenously prepared DFDBA is a cost effective, biocompatible material with osteogenic potential that can be used effectively in treating osseous defects of periapical lesions associated with non vital teeth.     

Bone regeneration: in vitro evaluation of the behaviour of osteoblast-like MG63 cells placed in contact with polylactic-co-glycolic acid, deproteinized bovine bone and demineralized freeze-dried bone allograft

Insufficient bone density of the alveolar crests, caused by loss of the dental elements, sometimes impedes the primary stability of an integrated bone implant. The techniques of bone regeneration allow to obtain a sufficient quantity of alveolar bone to permit the implant rehabilitation of the edentulous crests. Today several grafting materials are available and they have different characteristics, according to their structure, which influence the different behaviour of the grafting materials to the bone and the implant surface. The aim of this study is to evaluate the interaction between a human osteosarcoma MG63 cell line and three different biomaterials: polylactic-co-glycolic acid (PLAGA), deproteinized bovine bone and demineralised freeze-dried bone allograft (DFDBA). From this study a different behaviour emerges of the osteoblast-like MG63 cells in relation to the sublayer on which these cells were placed in culture. The results of the study, in fact, demonstrate that the most osteoconductive material of the three analysed is the DFDBA, followed by DPBB. On the contrary, the PLGA, because of its roughness, does not seem to represent a valid support for cell growth, and does not encourage any morphologic modification in tumor cells. Furthermore, deproteinized bovine bone shows a differentiating effect which could lead to hypothesise an osteoconductive capacity of this biomaterial. Further studies should be carried out with the aim of explaining the results obtained.     

Diagnosis and surgical treatment of defects in the wall of the orbit of children and adults using demineralized bone allografts

Accuracy of diagnosis defines the quality of treatment in patients with traumatic damage to eyelet walls. In this area, complex functional and anatomical breaches are typical and require full characterization of pathological changes in bone and soft tissue structures. A new plastic material with a high degree level of demineralization called “Perfoost” can be used to treat defects in the bones of the face of children and adults. In the present study, 79 patients with fractured eyelet walls were treated between 1999 and 2006 by grafting the defect wall with demineralized bone allografts. Grafts were applied from 2 days to 18 months after trauma. Magnetic resonance computer CT was used to check the realignment of allografts every 6 months after the reconstructive operation. The post-operative period of the observation was from 6 months to 7 years after the operation. Good or satisfactory results were obtained for 97.47 % of patients.     

Experimental and clinical study of the demineralized bone allografts manufactured in the tissue bank of CITO

The technology for producing demineralized bone allografts (DBA) with definable degree of demineralization and sterilization by high energy electron bean was developed in the tissue bank of Central Institute of Traumatology and Orthopaedics (CITO). The authors consider the technology to be one of the ways of producing demineralized bones. The results of the experiments show that time of demineralization process as well as the absorption dose of radioactive high energy electron beam change substantially mechanical toughness and osteoinductive properties of DBA. Mechanical properties of DBA were tested by the universal testing machine 'Zwick 1464'. Quantitative assessment of DBA osteoinductive properties resulted from the investigation of DBA samples in the culture of stromal precursor bone marrow cells and in the culture of human skin fibroblasts. Cloning efficiency of fibroblasts was considered as indecies of proliferative potential of stromal bone marrow cells, i.e. osteogenic precursor cells. The growth of the cell mass after definite time as well as the index of ³H-timidin marked cells within biological inductor were considered as indecies of proliferative potential of skin fibroblasts. The obtained results showed, that inductive properties of allografts improve when the degree of their demineralization increases whereas the dose of high energy electron radiation decreases. Mechanical toughness of DBA deteriorates when both degree of their demineralization and radiation doses increase. This emphasizes the importance of optimizing technological stages in DBA producing. Since 1998 DBA have been used in Russian clinics for bone plasty in traumatology and orthopaedics, maxilla-facial surgery, ophthalmology, and neurosurgery. The resulting analysis is based on case histories of 257 patients operated from March 1998 to July 2002. The majority of patients were children and teenagers of 3–18 years old with prime tumors, tumor-like and systemic inherited diseases of skeleton, post-traumatic complications. Observation periods were from 1.5 to 5 years and 10 months. Good and satisfactory results were obtained in 93.4% cases. This revised version was published online in July 2006 with corrections to the Cover Date.     

The in vitro chondrogenic response of limb-bud mesenchyme to a water-soluble fraction prepared from demineralized bone matrix

Demineralized adult bone matrix initiates de novo ectopic endochondral ossification 2-3 weeks following its intramuscular implantation into adult animals. This phenomenon appears to be similar, in some ways, to inductive cell-matrix interactions which regulate cartilage and bone formation during development. In the present study, we used embryonic chick limb-bud mesenchymal-cell cultures to bioassay extracts of demineralized bone matrix for chondrogenic activity. Guanidinium-chloride (4 M) extracts of demineralized bovine bone were dialyzed against buffers of decreasing ionic strength and then cold water. The cold-water-soluble fraction was found to stimulate chondrogenesis in intermediate-density limb-bud cell cultures (2.2 X 10(6) cells per 35-mm dish), as revealed by visual inspection with phase optics, toluidine-blue staining of fixed plates, and [35S] sulfate incorporation in the cell layer. Further fractionation of this material by anion-exchange, carbohydrate-affinity, and molecular-sieve chromotography produced a semipurified preparation possessing chondrogenic-stimulating activity at doses ranging from 3 to 10 micrograms/ml. The in vitro chondrogenic response of limb-bud mesenchymal cells was dose-dependent, required a minimal initial plating density of 2.08 X 10(5) cells/mm2 of culture dish, and developed gradually over 8-10 days. At an optimal dose of extract, a continuous exposure period of at least 2-3 days was necessary to produce detectable chondrogenic stimulation. In addition, the amount of cartilage formed following an 8-day exposure was markedly influenced by the culture 'age' of the mesenchymal cells (i.e., the time between plating and the start of treatment).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative study of alkaline phosphatase isoenzymes,bone histology,and skeletal radiography in dialysis bone disease.

Liver, intestinal, and bone alkaline phosphatase isoenzymes were measured using heat stability and L-phenylalanine inhibition techniques in 78 patients on intermittent haemodialysis. Fifty-five patients had abnormalities in one or more of the isoenzymes. Changes in bone and intestinal alkaline phosphatase activities seemed to be related and raised liver isoenzyme activity was associated with the development of liver disease. Abnormal histological and radiological findings were better correlated with bone alkaline phosphatase levels than with total alkaline phosphatase, and serial estimations of bone isoenzyme activity were useful in assessing the response of renal osteodystrophy to treatment with a vitamin D analogue. Serum alkaline phosphatase isoenzyme measurement provides another useful and non-invasive index for monitoring metabolic bone disease in patients with chronic renal failure.     

Distribution of bone inductive proteins in mineralized and demineralized extracellular matrix

Implantation of demineralized extracellular bone matrix results in new bone formation locally. Although the precise molecular mechanisms are not known, the reconstitution of matrix proteins less than 50,000 daltons with collagenous residue results in bone induction. The aim of the present investigation was to ascertain the distribution of the bone inductive protein(s) in various compartments of the tissue. A sequential extraction of mineralized bone matrix was employed: (1) 4 M guanidine HCl to extract proteins that are cell associated and not masked by mineral; (2) 0.5 M EDTA to dissolve the mineral phase; (3) 4 M guanidine HCl to reextract the collagenous matrix-associated proteins under dissociative conditions; (4) 4 M guanidine HCl containing 0.5 M EDTA to release any other residual proteins. This sequential method revealed that about 25% of total biological activity of bone induction is associated with first guanidine extraction, about 15% with the mineral phase and the rest of the activity is tightly associated with the collagenous matrix.     

Cancellous bone allograft seeded with human mesenchymal stromal cells: a potential good manufacturing practice-grade tool for the regeneration of bone defects

Background aimsCombining autologous bone precursor cells with cancellous bone allograft (CBA) offers an appealing strategy for skeletal regeneration. In this context, multipotent mesenchymal stromal cells (MSC) provide an excellent cell source because they are readily harvested from donors, expanded and differentiated in vitro. The aim of this study was to evaluate the proliferation, morphology, osteogenic differentiation and stem cell-related gene expression during static long-term ex vivo cultivation using human MSC and CBA under good manufacturing practice (GMP)-conforming conditions.MethodsMSC were isolated from healthy donors (n = 5) and cultivated on peracetic acid-sterilized CBA in the presence of 10% human platelet-rich plasma without osteogenic supplements. Total protein content, cell-specific alkaline phosphatase (ALP) activity and osteogenic marker gene expression levels were assessed. Stem cell-related gene expression was compared with MSC monolayer cultivation using microarray analysis. Furthermore, cellular distribution and morphology within the porous CBA were visualized by histology and scanning electron microscopy.ResultsEffective adhesion, spreading, proliferation and intercellular contact of human MSC within the pores of CBA were observed during the study (≤42 days). Cell-specific ALP activity peaked after 3 weeks of cultivation. Gene expression of early, intermediate and late osteogenic marker genes was detectable during long-term cultivation. Microarray-based annotation and biologic interaction network data analysis indicated that expression levels of genes encoding crucial differentiation-regulating proteins and extracellular matrix components involved in the process of osteogenesis were induced in CBA-cultivated MSC.ConclusionsMSC-vitalized CBA offers an attractive GMP-grade bone-filling material. Further research is warranted to evaluate its bone-healing potential in vivo.     

Clinical applications of mesenchymal stem cells in the treatment of fracture non-union and bone defects

Bone defects resulting from trauma or resorption, whether physiological or pathological, remain a major challenge in the management of patients. The limits of bone regeneration still result in many people never recovering fully their function and quality of life; with all the social, financial and psychological implications. The aim of this review is to present the current level of possible applications of stem cells and tissue engineering in bone repair. From animal models to human trials, the knowledge surrounding the use of mesenchymal stem cells in manipulating bone healing, where normal physiological procedures have failed, are presented in chronological order. The possibilities in clinical applications of mesenchymal stem cells are evident and exciting. The efficacy, including long-term, of such treatment options still requires further knowledge and appropriately conducted clinical trials, with adequate patient numbers. Once these techniques are properly mastered and perfected, the benefits to regenerative medicine will be immense.     

In vitro and in vivo evaluation of xenogeneic bone putty with the carrier of hydrogel derived from demineralized bone matrix

The demineralized bone matrix (DBM) putty is a traditional bone graft utilized to facilitate the repair and reconstruction of bone. Recent studies indicated the DBM putties with the various carriers were different in bone repairing ability. In order to prepare a kind of DBM putty with a good biocompatibility and bioactivity, the DBM gel was processed from the DBM and the feasibility as a carrier for the DBM putty was evaluated. After the bovine DBM gel was prepared, the BMPs content as well as the ability to promote osteogenic differentiation of MC3T3-E1 cells in vitro were investigated. Then the DBM putty was prepared and filled into the rat calvarial defect model to evaluate the bone repairing ability by micro-CT and histology. The result showed there was 2.953?±?0.054 ng BMP contained in per gram of the DBM gel. And the ALP production of MC3T3-E1 cells in the DBM gels group increased with prolonged culturing, the mineralized nodules formed in MC3T3-E1 cells on 14th day after co-culture. The putty prepared by DBM gel was easy to handle without loss of DBM particles at room temperature. In the rat calvarial bone defect experiment, histological observation showed more mature bone formed in the DBM putty group than that in the type I collagen group at 12 weeks, which indicated the bone putty prepared by DBM gel exhibited a better bone repair capability.