Impaired Bone Healing Research Articles

Do Inhibitors of Cyclooxygenase-2 Impair Bone Healing?

Do Inhibitors of Cyclooxygenase-2 Impair Bone Healing?

Study of the healing process after transplantation of pasteurized bone grafts in rabbits.

Different bone allografts (pasteurized, autoclaved, and frozen) were compared based on their osteoinductive properties. Our primary purpose was to examine the biologic qualities of pasteurized allografts, as pasteurization inactivates most viruses transmitted by transplantation. Frozen, pasteurized, and autoclaved allografts were packed into a standard defect of rabbit ulna. The animals were sacrificed at 2 and 4 weeks after surgery. The parts of bones with experimental defects were explored en bloc, and a roentgenogram was carried out. Ulna bone samples were then embedded in methyl-methacrylate. Roentgenograms showed that after 2 weeks, calluses were well-formed, but irregular in shape in all 3 types of allografts. After 4 weeks, the calluses were regular in shape in all but the autoclaved grafts. After 2 weeks, the healing processes had begun in the frozen and pasteurized grafts, with the reaching approximately the same stage, while in the autoclaved grafts these processes were not seen and the bone particles were surrounded by connective tissue without any changes. After 4 weeks, osteoinductive processes were very strong, with the first signs of complete bone remodeling at the bone edges of the defect in pasteurized and frozen allografts. The osteoinductive values of these 2 types were very high and similar. Autoclaved allografts, on the other hand, had very low osteoinductive values, as they were still at the very beginning of the healing process. Histomorphometric analysis revealed a significant difference in both newly formed osteoid thickness and osteoblast number per microm of bone surface in all experimental groups (P < 0.005). Values of osteoid thickness and osteoblast number were significantly higher in both frozen and pasteurized grafts when compared with the autoclaved ones (P < 0.005). Osteogenic properties of pasteurized bone allografts were preserved, and the allografts have been gradually replaced with newly formed bone. As such, pasteurized bone grafts from a bone bank have approximately the same biologic validity as frozen grafts, while autoclaved grafts impair bone healing.

Bone-resorbing activity from cholesterol-exposed macrophages due to enhanced expression of interleukin-1alpha.

The presence of cholesterol crystals, macrophages, and foreign giant cells has been associated with impaired bone healing of periapical lesions. Therefore, we investigated whether macrophages exposed to cholesterol crystals can release factors changing the activity of bone-resorbing osteoclasts. Mouse peritoneal macrophages treated with cholesterol crystals in vitro produced factor(s) that stimulated the release of 45Ca and 3H from mouse calvariae pre-labeled with 45Ca(CaCl2) or [3H]-proline, respectively. No bone-resorbing activity was released by epithelial cells, fibroblasts, or osteoblasts exposed to cholesterol crystals. Interleukin-1 receptor antagonist protein and antiserum neutralizing mouse interleukin-1alpha (IL-1alpha) inhibited 45Ca release induced by cholesterol-activated macrophages. The addition of cholesterol to the macrophages augmented the release of IL-1alpha protein and the expression of IL-1alpha mRNA. These findings indicate that frustrated phagocytosis by macrophages exposed to cholesterol crystals results in release of factors stimulating osteoclastic bone resorption, primarily due to increased transcription of the IL-1alpha gene.

Orthodontic spring guidance of bilateral mandibular distraction in rabbits

Although distraction osteogenesis can lengthen congenitally small mandibles, the distraction procedure can be difficult to control. To study the efficacy and safety of orthodontic spring guidance on bilateral mandibular distraction, an 8-mm anterior open bite was experimentally produced and corrected during bilateral mandibular distraction in rabbits. Orthodontic springs were attached to the anterior maxilla and mandible to redirect an ongoing distraction procedure. Sixteen rabbits underwent mandibular distraction: 6 rabbits received heavy springs (8 oz), 6 rabbits received light force springs (2 oz), and 4 rabbits served as control animals with anterior open bites without spring guidance. Nickel-titanium springs were applied during the last week of osseous distraction and the first week of consolidation. Distractors were left in place throughout a 2-month consolidation period. None of the animals developed fibrous union as a result of spring guidance. The 8-mm open bite did not close in the control group or in the light spring group after 2 weeks of spring wear or during the consolidation period. Heavy springs completely closed the experimental open bites within 2 weeks (P <.01, analysis of variance). Bite corrections did not change during the consolidation period. This study indicated that the addition of an orthodontic spring to a mandibular distraction procedure did not impair bone healing. With the distraction device in place, heavy spring forces redirected an ongoing mandibular distraction procedure and corrected an open bite, distraction side effect. Direct measurements, radiographic measurements, and tissue histologic factors described changes in segment position and shape of the distraction site. (Am J Orthod Dentofacial Orthop 2001;120:435-42)

Tissue reactions to particles of bone-substitute materials in intraosseous and heterotopic sites in rats: discrimination of osteoinduction, osteocompatibility, and inflammation

Two rat models were used to characterize tissue-specific reactions to particles of bone-substitute materials: one for osteocompatibility in a healing tibial wound and the other in a heterotopic, subcutaneous site. Small, unicortical tibial wounds in rats healed spontaneously, beginning with the rapid proliferation of intramedullary woven bone. That temporary bone was resorbed by osteoclasts and finally, the cortical wound was healed with lamellar bone and the medullary space was repopulated with marrow. When various particulate materials were implanted into fresh wounds, three types of reactions were observed. (1) Demineralized bone powder (DBP) and non-resorbable calcium phosphate (nrCP) were incorporated into the reactive medullary and cortical bone. (2) Polymethylmethacrylate (PMMA) particles were surrounded with a fibrous layer, but did not impair bone healing. (3) Polyethylene (PE) shards and resorbable calcium phosphates (rCPs) were inflammatory and inhibited osseous repair. Subcutaneous sites showed osteoinductive, fibrotic, or inflammatory responses to these materials. Only DBP induced endochondral osteogenesis subcutaneously. The nrCP evoked a fibrous reaction. In contrast, rCPs, PMMA, and PE shards generated inflammatory reactions with each particle being surrounded by fibrous tissue and large multinucleated giant cells. In conclusion, only DBP showed osteoinductive as well as osteocompatible properties. The nrCP was osteocompatible. The rCPs stimulated various degrees of inflammatory responses. PMMA was osteocompatible and did not interfere with the bone healing process. PE was not osteocompatible and generated foreign body reactions in both sites. Use of the two sites distinguishes osteoinductive, osteocompatible, and inflammatory properties of particles of bone-substitute materials.

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.

Promoted bone healing at a rabbit skull gap between autologous bone fragment and the surrounding intact bone with biodegradable microspheres containing transforming growth factor-beta1.

This study is a trial to promote repairing of the rabbit skull bone gap between an autologous bone flap and the intact bone with biodegradable gelatin microspheres containing transforming growth factor-beta1 (TGF-beta1). A 10-mm diameter bone defect was prepared in rabbit skulls by drilling out a bone flap of 6 mm in diameter. After a surrounding gap defect of 2 mm was created and treated with 0.5 microg of free TGF-beta1 and gelatin microspheres containing 0.5 microg of free TGF-beta1, the circular autologous bone flap was placed in the center. Significant bone healing at the gap defect was observed 3 weeks after implantation of the TGF-beta1-containing gelatin microspheres. The bone mineral density (BMD) was significantly higher than that of other experimental groups. On the contrary, when applied with free TGF-beta1, a fibrous tissue initially infiltrated into the gap defect, resulting in impairing bone healing. The tissue response was similar to that at the defect implanted with empty gelatin microspheres and TGF-beta1-free phosphate-buffered saline solution alone. There was more space in the gap-filling bone in the 16-week view than the 3-week view. It is possible that this was an intermediate step along the way toward normal healing and formation of cancellous bone. We conclude that gelatin microspheres containing TGF-beta1 show promise as an agent to promote bone regeneration of subcritical size defects between surgically positioned autologous bone flaps and surrounding host bone.

Parathyroid hormone-related protein analog RS-66271 is an effective therapy for impaired bone healing in rabbits on corticosteroid therapy

A new class of parathyroid hormone-related protein (PTHrP) analogs has been developed that causes a rapid gain in both trabecular and cortical bone in models of osteopenia. This study investigates the efficacy of the PTHrP analog, RS-66271 ([MAP 1–10] 22–31 hPTHrP(1-34)–NH 2), as systemic therapy for impaired bone healing in corticosteroid-treated rabbits. A 1 mm defect was created bilaterally in the ulnae of 30 rabbits. Delayed healing was induced by daily injections of prednisone (0.15 mg/kg) beginning 2 months prior to surgery and continuing until killing. Rabbits in the experimental group received daily subcutaneous injections of PTHrP analog RS-66271 (0.01 mg/kg) starting 1 day after surgery. Control animals received subcutaneous normal saline. At the 6 week timepoint, nine of ten ulnae from PTHrP-treated rabbits achieved radiographic union, whereas only two of ten limbs achieved union in control rabbits ( p < 0.01). In a separate part of the study, 20 animals (10 control, 10 RS-66271-treated) were killed when radiographic union was achieved bilaterally. In this portion of the study, all limbs in animals treated with PTHrP achieved union by 6 weeks. In the control animals that were allowed to heal for 10 weeks, only 20% of the limbs achieved radiographic union. In addition, ulnae in the PTHrP-analog-treated rabbits showed greater radiographic intensity (20%–40%), larger callus area (209% anteroposterior view, 417% lateral view) (mean area on AP radiographs: control, = 387 ± 276 mm 2; PTHrP analog, 1195 ± 408 mm 2), and greater stiffness (64%) and torque (87%) when compared with controls. RS-66271 was shown to be an effective therapy for preventing impaired bone healing caused by prednisone in a rabbit model.

Impact of nicotine on bone healing

A limited number of experimental animal studies and in vitro data confirm that nicotine impairs bone healing, diminishes osteoblast function, causes autogenous bone graft morbidity, and decreases graft biomechanical properties. Therefore, our long-term goal is to develop an effective therapy to reverse the adverse impact of nicotine from tobacco products. However, before accomplishing this goal, we had to develop an animal model. Our hypotheses were nicotine administration preceding and following autogenous bone grafting adversely affected autograft incorporation and depressed donor site healing in a characterized animal wound model. Hypothesis testing was accomplished in bilateral, 4-mm diameter parietal bone defects prepared in 60 Long-Evans rats (male, 35-day-old). A 4-mm diameter disk of donor bone was removed from the left parietal bone and placed in the contralateral defect. The donor site served as a spontaneously healing bone wound. The rats were partitioned equally among three doses of nicotine administered orally in the drinking water (12.5, 25, and 50 mg/L). For each dose, the duration and sequence of nicotine treatment followed four courses, including no nicotine and designated combinations of nicotine administration and abatement prior to and following osseous surgery. Experimental sites were recovered on 14 and 28 days postsurgery, responses quantitated, and data analyzed by analysis of variance and post hoc statistics (p < or = 0.05). We developed a convenient and effective osseous model, and the results validated our hypothesis that nicotine negatively impacts on bone healing.

Clinical and histologic observations of sites implanted with intraoral autologous bone grafts or allografts. 15 human case reports.

The cases reported in this paper were treated at 7 different clinical centers and present clinical and histologic observations from 15 patients and 21 human biopsies. The biopsies were taken from extraction sockets or dental implant sites which were grafted with either autologous intra-oral bone (6 sites), demineralized freeze-dried bone (DFDBA) (7 sites), or mineralized freeze-dried bone (MFDBA) (7 sites), or a combination of autologous bone, DFDBA and a barrier membrane (1 site). Six sites were grafted with DFDBA and augmented with expanded polytetrafluoroethylene (ePTFE) barrier membranes. Biopsies for histological evaluation were taken 4 to 13 months after implantation. A bone scoring system of 0 to 4 was used to evaluate the sections for dead implanted particles or the presence of vital bone. A bone score of 3 indicated the presence of dead implant material, blood vessels, islands of cartilage, osteoblasts, and new bone formation. A score of 4 indicated total replacement of the implanted material by the host bone. The average bone score for sites which received autologous bone was 2.33; for DFDBA sites, 0.98; and MFDBA was 0.18. The over-riding histologic characteristic of sites implanted with DFDBA or MFDBA was retention of non-vital graft particles within fibrous connective tissue. Biopsies taken adjacent to the host bed demonstrated incorporation of the allografts (osteoconduction). Sites grafted with autologous bone chips also demonstrated non-vital bone chips surrounded by vital host bone (osteoconduction). Sites which received barrier membranes did not appear to improve or impair bone healing of the augmented sites. Autologous bone chips harvested from within the oral cavity as well as allografts may serve as biologic fillers, but do not apparently contribute to osteoinduction. Autologous bone will eventually be resorbed and replaced by the host. DFDBA and MFDBA are resorbed very slowly and apparently do not contribute to osteoinduction. Allografts apparently are not resorbed by osteoclasts and therefore their continued use around dental implants is questioned.

Treatment of Nonunion with a Constant Direct Current: A Totally Implantable System

There is now sufficient basic research and clinical experience to establish that electrical stimulation produces osteogenesis. Furthermore, electrical stimulation significantly helps union where impaired bone healing exists. The implanted bone growth stimulator is one effective method of electrical stimulation. It can be used in a wide variety of problems: delayed union and nonunion of bones with or without chronic infection and in failed posterior spinal fusion. Successful treatment of congenital pseudarthrosis of the tibia has been encouraging. The implanted bone growth stimulator technique requires a simple operation with strict adherence to detail. There is minimal postoperative discomfort and a short hospital stay. The average time to union is 16 weeks. The Osteostim can be used in the presence of chronic infection and internal fixation. Above all, the technique does not require any cooperation from the patient. The implanted bone growth stimulator should be accepted as a method of treatment for delayed and nonunion of bones, as it is at least as effective as other more conventional methods of surgical treatment for this situation. It has been proved that electrical stimulation produces osteogenesis. Orthopedic surgeons should no longer be skeptical about this.

Donor marrow-derived cells as immunogens and targets for the immune response to bone and skin allografts.

The relative importance of donor marrow-derived cells in the immunogenicity of bone and skin allografts was compared. Radiation chimeras were created to have marrow-derived cells (MDCs) of a different genotype from their nonmarrow-derived cells (NMDCs). Such animals were used as donors of bone or skin for recipients chosen so that either the MDCs or the NMDCs of the graft would be incompatible. Immunogenicity was determined by measuring the recipient antibody response. The effect of the immune response on the bone graft (rejection) was determined by impaired bone healing. When MDCs alone were H-2 disparate with the recipient, bone grafts were immunogenic, and the bone graft healing was impaired. In contrast, skin grafts in the same combinations were immunogenic but were not rejected if the differences were only expressed on the MDCs of the graft. The role of NMDCs in all of these experiments was more difficult to interpret, but the results suggested that NMDCs are relatively unimportant for healing of bone grafts, although critical for rejection of skin grafts. We conclude that, unlike the situation with skin grafts, the major inducers and targets of the immune response to bone allografts are marrow derived.