Guided bone regeneration produced by new mineralized and reticulated collagen membranes in critical-sized rat calvarial defects

The aim of this study was to evaluate the bone regenerative effect of glutaraldehyde (GA) cross-linking on mineralized polyanionic collagen membranes in critical-sized defects on rat calvarias. Bone calvarial defects were induced in Wistar rats, which were then divided into five groups: a sham group; a control group, which received a commercial membrane; and GA, 25GA, and 75GA groups, which received one of three different polyanionic collagen membranes mineralized by 0, 25, or 75 hydroxyapatite cycles and then cross-linked by GA. Bone formation was evaluated based on digital radiography and computerized tomography. Histological analyses were performed 4 and 12 weeks after the surgical procedure to observe bone formation, membrane resorption, and fibrous tissue surrounding the membranes. Measurement of myeloperoxidase activity, tumor necrosis factor alpha, and interleukin 1beta production was performed 24 h after surgery. The percentage of new bone formation in the GA, 25GA, and 75GA groups was higher compared with the control and sham groups. In the GA and 25 GA groups, the membranes were still in place and were contained in a thick fibrous capsule after 12 weeks. No significant difference was found among the groups regarding myeloperoxidase activity and interleukin 1beta levels, although the GA, 25GA, and 75GA groups presented decreased levels of tumor necrosis factor alpha compared with the control group. These new GA cross-linked membranes accelerated bone healing of the calvarium defects and did not induce inflammation. In addition, unlike the control membrane, the experimental membranes were not absorbed during the analyzed period, so they may offer advantages in large bone defects where prolonged membrane barrier functions are desirable.     

The effect of temperature exposure during shipment on a commercially available demineralized bone matrix putty

During August and September of 2013, temperature data loggers were shipped to and from an AATB accredited and FDA registered allograft tissue processing facility in Belgrade, MT (Bacterin International, Inc.) to five warm climate cities (Dallas, TX, El Paso, TX, New Orleans, LA, Phoenix, AZ, and Tampa, FL). Shipping data acquired from 72 independent shipments were analyzed to generate an assessment of temperature exposure, shipment times, and shipping event durations experienced during routine distribution. Overall the packages experienced an average temperature of 26.2 ± 2.3 °C which mirrored the average external ambient temperature of 25.8 ± 3.0 °C. However, temperature spikes above 40 °C were frequently observed. The data from the model shipments were extrapolated to provide a worst-case high temperature spike of 52.9 °C for 12 h and 14 min. Multiple lots of a commercially available demineralized bone matrix (DBM) putty (OsteoSelect^® DBM Putty) were subjected to continuous heating at 50 °C, to multiple worst-case temperature spikes, and to multiple freeze–thaw cycles to assess the effects of these temperature extremes on the handling and osteoinductivity of the allograft tissue. Five weeks of continuous exposure to 50 °C and 12 simulated worst-case one-way shipments did not adversely affect the handling characteristics or the in vivo osteoinductivity of the product.     

Local antibiotic delivery with demineralized bone matrix

A method of care for these infected nonunions is prolonged intravenous systemic antibiotic treatment and implantation of methyl methacrylate antibiotic carrier beads to delivery high local doses of antibiotics. This method requires a second surgery to remove the beads once the infection has cleared. Recent studies have investigated the use of biodegradable materials that have been impregnated with antibiotics as tools to treat bone infections. In the present study, human demineralized bone matrix (DBM) was investigated for its ability to be loaded with an antibiotic. The data presented herein demonstrates that this osteoinductive and biodegradable material can be loaded with gentamicin and release clinically relevant levels of the drug for at least 13 days in vitro. This study also demonstrates that the antibiotic loaded onto the graft has no adverse effects on the osteoinductive nature of the DBM as measured in vitro and in vivo. This bone void filler may represent a promising option for local antibiotic delivery in orthopedic applications.     

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.     

An overview on bone protein extract as the new generation of demineralized bone matrix

Bone protein extract is regarded as the new generation of demineralized bone matrix. The aim of this paper is to describe and characterize the properties of demineralized bone matrix and its new generation product in addition to its application in animal and human studies. Bone protein extract has features of osteoconductivity, osteoinductivity and osteogenicity, which originate from its unique and precise processing. It has exhibited powerful bone formation capacity both in animal experiments and in clinical trials by providing an optimal microenvironment for osteogenesis. Furthermore, not only does it have excellent biocompatibility, it also has good compatibility with other implant materials, helping it bridge the host and implanted materials. Bone protein extract could be a promising alternative for demineralized bone matrix as a bone graft substitute.     

Preparation and biocompatibility of demineralized bone matrix/sodium alginate putty

Demineralized bone matrix (DBM) powder is widely used for bone regeneration due to its osteoinductivity and osteoconductivity. However, difficulties with handling, tendency to migrate from graft sites and lack of stability after surgery sometimes limit the clinical utility of this material. In this work, the possibility of using sodium alginate (ALG) carrier to deliver DBM powder was assessed. DBM–ALG putty with the DBM:ALG weight ratio of 5:5, 6:4, 7:3, 8:2 were prepared, respectively. The properties of the formed composite, including discrete degree, washout property, pH, equilibrium swelling as well as cytotoxicity in vivo, were adopted to ascertain the optimal ratio of DBM and ALG. The discrete diameter increased from 1.25 cm (5:5) to 2.08 cm (8:2) with the increase of DBM content. There was significant difference between the 8:2 group and the other groups in discrete diameter. The ratio of DBM had a significant effect on the swelling value. The pH of composites showed an increase trend with the DBM ratio’s increase, when the ratio reached 7:3, the pH (7.22) was approximately equal to the body fluid. The proliferation of MC3T3-E1 cells was inhibited in the 5:5, 6:4 and 7:3 groups, while a slightly increased in the 8:2 group. The DBM–ALG with the optimal ratio of 7:3 was confirmed based on the results of the above mentioned. The histocompatibility of DBM–ALG (7:3) was examined using a rat model in which the materials were implanted subcutaneously, compared with the polyethylene, ALG and DBM. The study in vivo showed DBM–ALG (7:3) had a lower inflammatory response than DBM, a higher vascularization than ALG. The osteoinduction of DBM–ALG (7:3) was evaluated by co-culturing with MC3T3-E1 in vitro, compared with the DMEM, ALG and DBM. The results indicated calcification area in the DBM–ALG group was similar to that in the DBM group, larger than ALG group and DMEM group. The DBM–ALG (7:3) putty is promising as a directly injectable graft for repair of bone defect.     

Vitreous cryopreservation of tissue engineered bone composed of bone marrow mesenchymal stem cells and partially demineralized bone matrix

Cryopreservation of tissue engineered products by maintaining their structure and function is a prerequisite for large-scale clinical applications. In this study, we examined the feasibility of cryopreservation of tissue engineered bone (TEB) composed of osteo-induced canine bone marrow mesenchymal stem cells (cBMSCs) and partially demineralized bone matrix (pDBM) scaffold by vitrification. A novel vitreous solution named as VS442 containing 40% dimethyl-sulfoxide (DMSO), 40% EuroCollins (EC) solution and 20% basic culture medium (BCM) was developed. After being cultured in vitro for 8 days, cell/scaffold complex in VS442 was subjected to vitreous preservation for 7 days and 3 months, respectively. Cell viability, proliferation and osteogenic differentiation of cBMSCs in TEB after vitreous cryopreservation were examined with parallel comparisons being made with those cryopreserved in VS55 vitreous solution. Compared with that cryopreserved in VS55, cell viability and subsequent proliferative ability of TEB in VS442 after being rewarmed were significantly higher as detected by live/dead staining and DNA assay. The level of alkaline phosphatase (ALP) expression and osteocalcin (OCN) deposition in VS442 preserved TEB was also higher than those in the VS55 group since 3 days post-rewarm. Both cell viability and osteogenic capability of the VS55 group were found to be declined to a negligible level within 15 days post-rewarm. Furthermore, it was observed that extending the preservation of TEB in VS442 to 3 months did not render any significant effect on its survival and osteogenic potential. Thus, the newly developed VS442 vitreous solution was demonstrated to be more efficient in maintaining cellular viability and osteogenic function for vitreous cryopreservation of TEB over VS55.     

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)     

Effect of bupivacaine on muscle tissues and new bone formation induced by demineralized bone matrix gelatin.

Heterotopic bone formation induced by demineralized bone matrix gelatin (BMG) in bupivacaine-HCl-treated skeletal muscle was examined histologically. BMG was obtained by dehydrating diaphyseal shafts of femora and tibiae of male, 4-week-old Sprague-Dawley (SD) rats, cutting it into chips, and demineralizing and extracting the chips with various solutions. The BMG was implanted into the rectus abdominis muscle of male, 5-week-old SD rats, bupivacaine-HCl was injected at the same site, and the resulting plaques of tissues were examined histologically on days 5, 10, 15 and 20 after BMG implantation. Heterotopic bone formation occurred in all animals. The bupivacaine-treated group had more degenerated and injured muscle fibers, and more osteocytes than the control group. Electron microscopy showed that the basement membrane of muscle fibers was discontinuous and that many mononucleated cells resembling activated satellite cells were present on day 5. Many fibroblasts, undifferentiated mesenchymal cells and myogenic cells were seen in the area around the BMG. In new bones there were few osteocytes on day 10, but their numbers were increased on days 15 and 20 after implantation, especially in the bupivacaine-treated group. The population of osteocytes that increased rapidly may have included mononucleated cells similar to activated satellite cells.     

Transport distraction osteogenesis: a new method to heal adult calvarial defects

Popularized by Gavril Ilizarov in the 1960s, monofocal distraction osteogenesis has become a well-established method of endogenous bone engineering. This revolutionary surgical technique has significantly augmented the available reconstructive orthopedic and craniomaxillofacial procedures. Bifocal distraction osteogenesis, or bone transportation, is a modification of monofocal distraction that involves moving a free segment of living bone to fill an intercalary bone defect. Bifocal distraction has been applied successfully to reconstruct complex mandibular and long bone defects. Because traumatic or postsurgical calvarial defects do not spontaneously heal in humans older than 18 to 24 months of age, we hypothesized that bifocal distraction osteogenesis could be applied to the skull to close critical size calvarial defects. Critical size (15 x 15 mm) calvarial defects were created in eight New Zealand White rabbits. Next, a 15-mm x 10-mm calvarial box osteotomy was created just anterior to the skull defect. This osteotomy created a free bone segment that could be transported. A custom-made transport distraction device was fixed into place and the skin incision was closed. After a 4-day latency period, the distraction device was activated (0.5 mm once daily for 30 days) in seven animals; the distraction device in one animal was not activated and served as a control. All animals underwent 30 days of consolidation and were then killed. Radiographs and computed tomographic scans were performed at the following time points: end of latency period (postoperative day 4), mid-distraction (postoperative day 19), and end of consolidation period (postoperative day 64). Gross and histologic analysis was performed to evaluate the quality of the bony regenerate. The control animal healed with a fibrous union. Complete closure of the skull defects was observed in five of seven rabbits at the end of the consolidation period. One animal was removed from the study because of an early loosening of the distraction device, and one was removed because of device failure. Of the remaining five animals that completed the distraction protocol, radiographs and computerized tomographic scans showed successful ossification in all five rabbits at the end of the consolidation period. This study suggests that transport distraction osteogenesis is a promising technique that may be applied to a variety of commonly encountered craniofacial problems such as nonhealing calvarial defects.     

Dynamic compression promotes proliferation and neovascular networks of endothelial progenitor cells in demineralized bone matrix scaffold seed

Neovascularization is required for bone formation and successful fracture healing. In the process of neovascularization, endothelial progenitor cells (EPCs) play an important role and finish vascular repair through reendothelialization to promote successful fracture healing. In this study, we found that dynamic compression can promote the proliferation and capillary-like tube formation of EPCs in the demineralized bone matrix (DBM) scaffold seed. EPCs isolated from the bone marrow of rats have been cultured in DBM scaffolds before dynamic compression and then seeded in the DBM scaffolds under dynamic conditions. The cells/scaffold constructs were subjected to cyclic compression with 5% strain and at 1 Hz for 4 h/day for 7 consecutive days. By using MTT and real-time PCR, we found that dynamic compression can significantly induce the proliferation of EPCs in three-dimensional culture with an even distribution of cells onto DBM scaffolds. Both in vitro and in vivo, the tube formation assays in the scaffolds showed that the loaded EPCs formed significant tube-like structures. These findings suggest that dynamic compression promoted the vasculogenic activities of EPCs seeded in the scaffolds, which would benefit large bone defect tissue engineering.     

Functional heterogeneity of osteoclasts: matrix metalloproteinases participate in osteoclastic resorption of calvarial bone but not in resorption of long bone.

Data in the literature suggest that site-specific differences exist in the skeleton with respect to digestion of bone by osteoclasts. Therefore, we investigated whether bone resorption by calvarial osteoclasts (intramembranous bone) differs from resorption by long bone osteoclasts (endochondral bone). The involvement of two major classes of proteolytic enzymes, the cysteine proteinases (CPs) and matrix metalloproteinases (MMPs), was studied by analyzing the effects of selective low molecular weight inhibitors of these enzymes on bone resorption. Mouse tissue explants (calvariae and long bones) as well as rabbit osteoclasts, which had been isolated from both skeletal sites and subsequently seeded on bone slices, were cultured in the presence of inhibitors and resorption was analyzed. The activity of the CP cathepsins B and K and of MMPs was determined biochemically (CPs and MMPs) and enzyme histochemically (CPs) in explants and isolated osteoclasts. We show that osteoclastic resorption of calvarial bone depends on activity of both CPs and MMPs, whereas long bone resorption depends on CPs, but not on the activity of MMPs. Furthermore, significantly higher levels of cathepsin B and cathepsin K activities were expressed by long bone osteoclasts than by calvarial osteoclasts. Resorption of slices of bovine skull or cortical bone by osteoclasts isolated from long bones was not affected by MMP inhibitors, whereas resorption by calvarial osteoclasts was inhibited. Inhibition of CP activity affected the resorption by the two populations of osteoclasts in a similar way. We conclude that this is the first report to show that significant differences exist between osteoclasts of calvariae and long bones with respect to their bone resorbing activities. Resorption by calvarial osteoclasts depends on the activity of CPs and MMPs, whereas resorption by long bone osteoclasts depends primarily on the activity of CPs. We hypothesize that functionally different subpopulations of osteoclasts, such as those described here, originate from different sets of progenitors.     

Long bone osteoclasts display an augmented osteoclast phenotype compared to calvarial osteoclasts

Osteoclasts are multinucleated cells specialized in degrading bone and characterized by high expression of the enzymes tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CtsK). Recent studies show that osteoclasts exhibit phenotypic differences depending on their anatomical site of action.Using immunohistochemistry, RT-qPCR, FPLC chromatography and immunoblotting, we compared TRAP expression in calvaria and long bone. TRAP protein and enzyme activity levels were higher in long bones compared to calvaria. In addition, proteolytic processing of TRAP was more extensive in long bones than calvaria which correlated with higher cysteine proteinase activity and protein expression of CtsK. These two types of bones also exhibited a differential expression of monomeric TRAP and CtsK isoforms. Analysis of CtsK^−/− mice revealed that CtsK is involved in proteolytic processing of TRAP in calvaria. Moreover, long bone osteoclasts exhibited higher expression of not only TRAP and CtsK but also of the membrane markers CD68 and CD163.The results suggest that long bone osteoclasts display an augmented osteoclastic phenotype with stronger expression of both membranous and secreted osteoclast proteins.