Demineralized bone matrix as a biological scaffold for bone repair.

Experimental models were created in rat fibula to represent impaired bone healing so that biological deficiencies that cause bone repair to fail or to be delayed may be investigated. These models consist of a 4-mm-long segmental defect, created in rat fibula by osteotomy, and fitted with a 7-mm-long tubular specimen of demineralized bone matrix (DBM) over the cut ends of the fibula. The experiments in this study involved various modifications of the DBM scaffold designed to reduce its osteoinductive activity: steam sterilization (sDBM), ethylene oxide sterilization (eoDBM), trypsin digestion (tDBM), and guanidine hydrochloride extraction (gDBM). Bone healing was evaluated by bending rigidity of the fibula and mineral content of the repair site at 7 weeks post-surgery. The sDBM scaffolds resorbed completely by 7 weeks and hence this model was a nonhealing negative control. Rigidities in the unmodified DBM and tDBM groups were comparable, whereas in the gDBM and eoDBM groups it was significantly reduced. Histologically, in the 4-mm defects repaired with unmodified DBM, direct and endochondral bone formation in the scaffold and the defect resulted in a neocortex consisting of woven and lamellar bone uniting the broken bone by 7 weeks post-surgery. We conclude that the eoDBM and gDBM groups represent failure or delay of the bone repair process when compared with the unmodified DBM group in which the process is analogous to normal bone healing.

Repair of segmental bone defects in the rat: an experimental model of human fracture healing.

Bone repair models in animals may be considered relevant to human fracture healing to the extent that the sequence of events in the repair process in the model reflect the human fracture healing sequence. In the present study, the relevance of a recently developed segmental defect model in rat fibula to human fracture healing was investigated by evaluating temporal progression of rigidity of the fibula, mineral content of the repair site, and histological changes. In this model, a surgically created 2-mm-long defect was grafted with a 5-mm-long tubular specimen of demineralized bone matrix (DBM) by inserting it over the cut ends of the fibula. The temporal increase in rigidity of the healing fibula demonstrated a pattern similar to biomechanical healing curves measured in human fracture healing. This pattern was characterized by a short phase of rapidly rising rigidity during weeks 4-7 after surgery, associated with a sharp increase in the mineral content of the repair tissue. This was preceded by a phase of nearly zero rigidity and followed by a phase of slow rate of increase approaching a plateau. Histologically, chondroblastic and osteoblastic blastema originating from extraskeletal and subperiosteal (near fibula-graft junction) regions, infiltrated the DBM graft during the first 2 weeks. The DBM graft assumed the role of a "bridging callus." By weeks 6-8, most of the DBM was converted to new woven and trabecular bone with maximal osteoblastic activity and minimal endochondral ossification. Medullary callus formation started with direct new bone formation adjacent to the cortical and endosteal surfaces in the defect and undifferentiated cells in the center of the defect at 3 weeks. The usual bone repair process in rodents was altered by the presence of the DBM graft to recapitulate the sequential stages of human fracture healing, including the formation of a medullary callus, union with woven and lamellar bone, and recreation of the medullary canal.

Role of TGF-beta1 in platelet-mediated cardioprotection during ischemia-reperfusion in isolated rat hearts.

Platelets protect myocardium against ischemia-reperfusion injury. This study examined the role of platelet-derived TGF-beta1 in cardioprotection during ischemia-reperfusion. Isolated Sprague Dawley rat hearts were perfused with K-H buffer and subjected to 25 min of global ischemia followed by 30 min of reperfusion. Ischemia-reperfusion resulted in myocardial dysfunction indicated by increase in CPP and LVEDP, and decrease in dLVP. Perfusion of hearts with washed platelets or supernatant of aggregated platelets attenuated (P < 0.01) of myocardial dysfunction following ischemia-reperfusion. Ischemia-reperfusion resulted in a decrease in myocardial TGF-beta1 determined by immunohistochemistry. ELISA showed an increase in latent TGF-beta1, but a decrease in active TGF-beta1. Perfusion of hearts with platelets or aggregated platelet supernatant preserved myocardial TGF-beta1 content upon ischemia-reperfusion. Perfusion of hearts with recombinant TGF-beta1 also resulted in cardioprotection following ischemia-reperfusion qualitatively similar to that observed with platelets or aggregated platelet supernatants. RT-PCR analysis showed an increase in myocardial TGF-beta1 mRNA following ischemia-reperfusion. These observations indicate that platelets protect the myocardium against ischemia-reperfusion-mediated dysfunction at least in part by releasing TGF-beta1. Increase in both TGF-beta1 mRNA and latent TGF-beta1 does not indicate a defect in the translation of mRNA. Reduction in myocardial TGF-beta1 following ischemia-reperfusion suggests a defect in the conversion of latent TGF-beta1 to active TGF-beta1.

Mineralization and pH relationships in healing skeletal defects grafted with demineralized bone matrix.

Early studies had indicated that tissue repair is initially associated with a lower than normal serum pH that later becomes more alkaline. To determine how tissue pH may affect skeletal healing and mineralization, we used a rat skeletal repair model consisting of a long bone segmental defect grafted with acid-demineralized bone matrix (DBM), a biomaterial possessing both osteoinductive and osteoconductive repair properties. In this study, femoral and tibial diaphyses from young adult Sprague Dawley rats were cut into cylinders approximately 0.5 cm in length, demineralized in acid, perforated to accommodate a needle-type combination pH microelectrode, and grafted around a 0.3-cm-long diaphyseal fibula defect. The pH of repair tissues was recorded at various time intervals up to 28 days postgrafting. Healing and mineralization were monitored histologically and by the ash and calcium content of repair tissues. During the early healing phase, tissue pH was lower than normal serum pH, presumably because of an accumulation of acidic metabolites in tissue fluids. Subsequent pH increases to more alkaline values were accompanied by a rapid mineral deposition phase and a later phase characterized by a slow, gradual increase in tissue calcium content. The results of this study support previous observations suggesting that the pH of repair tissue fluids may play a regulatory role in the healing and mineralization of bone.

Calcification of implanted vascular tissues associated with elastin in an experimental animal model.

We have previously studied the process of calcification in bioprosthetic porcine heart valves crosslinked with glutaraldehyde. Observations using light microscopy had indicated that calcification of elastic fibers occurs in implanted heart valves, in addition to calcification associated with collagen fibers. To determine the contribution of elastin to the process of calcification, small pieces of rabbit aorta were cross-linked with 0.2% glutaraldehyde, rinsed in buffer, and implanted subcutaneously in young adult male rats. Cross-linked jugular vein implants were included as controls. After an implantation period of 1 month or longer, we observed many areas of calcification in the aortic media associated with elastin and fewer such areas associated with collagen. The elastin-rich aortic tissues accumulated more calcium than venous tissues. Calcium deposits appeared similar in both allogenic and xenogeneic implants. Calcified areas viewed under the electron microscope included intercellular nonfibrous material. Calcified areas involved predominantly the outer layers of elastic fibers. Calcific deposits included needle-like crystals of hydroxyapatite but often consisted of an amorphous flocculant material surrounded by crystals. The close spatial relationship of hydroxyapatite crystals and elastic membranes seen in this study may be relevant to the initiation of dystrophic calcification in glutaraldehyde cross-linked aortic grafts.

Electric field stimulation of human osteosarcoma-derived cells: a dose-response study.

In vitro electrical stimulation of human osteosarcoma-derived cells resulted in increased cell adherence and current directed cell migration. We have developed an electrical exposure system in which two steel electrodes imbedded in media-based agar, poured in a standard culture dish, are used to apply electric field signals to cells in culture without ion contamination from the electrodes. The cells were exposed to a 100 Hz pulsed DC electric signal at peak field strengths of 1, 10, 100, and 625 mV/cm in the culture media. The data showed no change in cell adherence at 1 and 10 mV/cm, an increase in adherence at 100 mV/cm, and a decrease in both adherence and cell proliferation at 625 mV/cm. Electric field stimulation in vivo has been found useful in accelerating the healing of fractures and non-unions, and the repair of surgical and cancer-related skeletal defects.

Treatment of nonunion by percutaneous injection of bone marrow and demineralized bone matrix. An experimental study in dogs.

Successful treatment of nonunited fractures remains a major clinical challenge. Because bone marrow and demineralized bone matrix (DBM) are capable of stimulating osteogenesis, experiments were designed to test the effectiveness of bone marrow or DBM or both when injected percutaneously into a canine nonunion model. Six-millimeter segmental defects were created in the midtibial diaphysis of 24 adult mongrel dogs and held distracted by external fixation. For comparative purposes, a 0.5-mm osteotomy was created in five dogs. Five weeks later, the 6-mm defects were injected with either saline, autogeneic marrow, DBM powder, a composite of bone marrow and DBM, or treated by open grafting techniques with autogenic cancellous bone. Healing of the defect was evaluated roentgenographically, biomechanically (three-point bending), histologically, and biochemically 13 weeks postsurgery. Marrow and DBM stimulated defect healing. However, the combination of bone marrow with DBM produced a synergistic response in the defect, which was greater than the sum of either marrow or DBM alone. Healing in the composite-grafted dogs was comparable to those treated by standard cancellous bone grafting. These data suggest that percutaneous injection of bone marrow and DBM may be a potential alternative that offers numerous advantages over standard open grafting techniques in the treatment of fractures with nonunited defects.

Healing of a large nonossifying fibroma after grafting with bone matrix and marrow. A case report.

Healing of a large tibial nonossifying fibroma in a 12-year-old girl occurred following excision, curettage, and filling of the bony defect with human demineralized bone matrix powder mixed with a small quantity of the patient's bone marrow. The use of this graft composite was successful in the treatment of the lesion and reduced the potential risk and morbidity associated with pediatric orthopedic surgical procedures with autogeneic iliac crest and other bone grafts.

Quantitative roentgenographic densitometry for assessing fracture healing.

A noninvasive method was developed to assess fracture healing using optical densitometric methodology. Photometric measurements of osseous tissue density were based on illuminance or intensity of light, transmitted through standard roentgenograms. The method was tested in 6-mm tibial segmental defects and single-cut osteotomy defects in adult mongrel dogs. The lowest measureable bone density in the defect was compared to bending rigidity of the involved extremity. For both types of defect, an exponential relationship was found between the densitometric evaluation of the healing fracture gap and bending rigidity. This method proved superior to a roentgenographic scoring method and is capable of detecting small differences in mineral content using standard roentgenograms. The high correlation between densitometric evaluation and bone rigidity indicates this method has potential for use in the in vivo assessment of fracture healing.

Osteogenesis in cranial defects and diffusion chambers. Comparison in rabbits of bone matrix, marrow, and collagen implants.

In rabbits, we compared calcification and bone formation by bone marrow, acid-demineralized bone matrix and glutaraldehyde-cross-linked Type I collagen implanted in intramuscular diffusion chambers or in trephine skull defects. The rabbits were killed 4 weeks postimplantation and calcification and osteogenesis were evaluated radiographically and histologically, and by calcium and alkaline phosphatase assays. Bone marrow produced bone and fibrous tissue within the chambers and had high alkaline phosphatase levels. Bone matrix in chambers with intact filters failed to induce bone formation within and outside the chambers, while glutaraldehyde-cross-linked collagen produced only scant calcific deposits following implantation in either diffusion chambers or skull defects. Central areas of skull defects implanted with bone marrow were partially repaired with new bone and had high calcium and alkaline phosphatase levels, but not as high as defects implanted with demineralized bone matrix.

Skeletal repair in the aged: a preliminary study in rabbits.

Bone mass loss associated with aging can lead to osteoporosis and multiple bone fractures with impaired healing requiring prolonged hospitalization and costly medical care. We have used an experimental implantation model to test the ability of old animals to form new bone. Bone repair inducers, consisting of demineralized bone matrix (DBM), bone marrow, and collagen, were implanted in the abdominal wall muscles of 1-month and 16-month old rabbits. DBM contains a bone morphogenetic protein (BMP) that induces the differentiation of primitive mesenchymal cells into bone producing cells. The stromal cells of bone marrow can differentiate into osteoblasts after implantation, while collagen could serve as a calcification nucleus or framework for new tissue formation. Animals were killed 4 to 6 weeks after implantation. Implants were X-rayed, examined histologically, and analyzed for water content, calcium, and alkaline phosphatase. Only the implants of bone marrow enclosed in filter chambers (0.45 micron pore diameter) were associated with bone formation. Intramuscular implants of DBM and bone marrow in the old animals induced the formation of new bone but contained less calcium and lower levels of alkaline phosphatase than implants in the young animals. Collagen implants were resorbed and failed to induce bone formation or calcify. The results indicate that formation of new bone, under the conditions of this study, is reduced with aging.

Ectopic bone formation is enhanced in senescent animals implanted with embryonic cells.

Ectopic bone formation induced by the subcutaneous implantation of demineralized bone matrix (DBM) is very significantly reduced in older Fischer 344 rats. Cells originating from calvaria of 20-day-old embryo donors were introduced into cylinders of DBM sealed at the ends with a Millipore filter or collagen sponges prior to subcutaneous implantation. Cells within the chambers had access to vascular channels that could penetrate through the interstices of the DBM. After four weeks of implantation in 26-month-old rats, the cylinders were full of bone. This bone was assessed by histologic techniques, by calcium and bone gamma-carboxyglutamic acid (gla) protein (BGP) concentrations, and by alkaline phosphatase activity. Cylinders to which no cells were added produced no bone. Bone marrow cells enclosed in similar cylinders or injected weekly at the implantation site also enhanced new bone formation but to a much lesser extent. Embryonic muscle cells formed large amounts of cartilage and less bone. Fibroblasts were inactive in this system. Prior treatment of the DBM with trypsin inhibited the myoblast response but not that of calvaria cells.

Biochemical differences between dystrophic calcification of cross-linked collagen implants and mineralization during bone induction.

Ectopic calcification of diseased tissues or around prosthetic implants can lead to serious disability. Therefore, calcification of implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen was compared with mineralization induced by demineralized bone matrix (DBM). Whereas implants of DBM accumulated large amounts of calcium and a bone-specific gamma-carboxyglutamic acid protein (BGP or osteocalcin) following implantation in both young and older rats, implants of cross-linked pericardium calcified with only traces of BGP. Glutaraldehyde-cross-linked DBM failed to calcify after implantation in 8-month-old rats for 2-16 weeks. Implants of cross-linked type I collagen exhibited small calcific deposits 2 weeks postimplantation but calcium content eventually dropped to levels equal to those of soft tissues as the implants were resorbed. The calcium content of DBM implanted in 1- and 8-month-old rats reached comparable levels after 4 weeks, but the BGP content was approximately twice as high in the younger animals than in the older ones. Glutaraldehyde-cross-linked implants of DBM, tendon, and cartilage calcified significantly in young but not in old animals. This form of dystrophic calcification was associated with only trace amounts of BGP. Alkaline phosphatase activity was high in implants of DBM and undetectable in implants of cross-linked collagenous tissues. These results show that implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen calcify to different extents depending upon their origin and the age of the host, and that the mechanism of dystrophic calcification differs significantly from the process of mineralization associated with bone induction as reflected by alkaline phosphatase activity and BGP accumulation.

Chemically modified collagen: a natural biomaterial for tissue replacement.

Glutaraldehyde crosslinking of native or reconstituted collagen fibrils and tissues rich in collagen significantly reduces biodegradation. Other aldehydes are less efficient than glutaraldehyde in generating chemically, biologically, and thermally stable crosslinks. Tissues crosslinked with glutaraldehyde retain many of the viscoelastic properties of the native collagen fibrillar network which render them suitable for bioprostheses. Implants of collagenous materials crosslinked with glutaraldehyde are subject long-term to calcification, biodegradation, and low-grade immune reactions. We have attempted to overcome these problems by enhancing crosslinking through bridging of activated carboxyl groups with diamines and using glutaraldehyde to crosslink the epsilon-NH2 groups in collagen and the unreacted amines introduced by aliphatic diamines. This crosslinking reduces tissue degradation and nearly eliminates humoral antibody induction. Covalent binding of diphosphonates, specifically 3-amino-1-hydroxypropane-1, 1-diphosphonic acid (3-APD), and chondroitin sulfate to collagen or to the crosslink-enhanced collagen network reduces its potential for calcification. Platelet aggregation is also reduced by glutaraldehyde crosslinking and nearly eliminated by the covalent binding of chondroitin sulfate to collagen. The cytotoxicity of residual glutaraldehyde--leaching through the interstices of the collagen fibrils or the tissue matrix--and of reactive aldehydes associated with the bound polymeric glutaraldehyde can be minimized by neutralization and thorough rinsing after crosslinking and storage in a nontoxic bacteriostatic solution.

A randomized study of two preparations for large bowel radiology.

A randomized study was carried out in 195 out-patients, due to undergo large bowel radiological examination, to compare the efficacy, patient acceptance and incidence of side-effects of a commercially-available bowel evacuant kit (magnesium citrate oral solution, phenolphthalein tablets and a bisacodyl suppository) and castor oil with enemas. Overall evaluation by a radiologist of large bowel preparation, based on post-barium evacuation X-rays, was satisfactory in more than 98% of patients while bowel cleanliness, as determined by the ability to detect a 1 cm lesion, was adequate in 95% of patients using either preparation. Patient acceptance was in favour of the commercial preparation in that fewer patients using it found the procedure uncomfortable or indicated a preference for another evacuant than did those prepared with castor oil and enemas.