Regeneration of the skeleton by recombinant human bone morphogenetic proteins

Recombinant human bone morphogenetic proteins (rhBMPs) have past a long journey in human orthopaedic surgery during the last 15 years. From the first reports of the use of rhBMPs in hostile environments such as critically-sized bone defects, avascular femoral head necrosis, unstable thoracolumbar vertebral fractures, instability between the atlas and axis due to rheumatoid arthritis; over the use for nonunions of long bones and the scaphoid, reconstructive and revision surgeries of the hip, acute fractures, allograft nonunions, congenital pseudarthrosis, and various approaches of lumbar and cervical spine fusions, rhBMPs overgrow to a safe and reliable device in the treatment of open tibial shaft fractures, nonunions of long bone fractures, anterior lumbar interbody fusion and revision posterolateral lumbar fusions. Systematic review of the published literature of rhBMPs is presented.     

Treatment of infected tibial nonunion with bone defect using central bone grafting technique

Treatment of infected tibial nonunion with bone defect represents a challenge for every orthopaedic surgeon. Various methods of treatment have been described for nonunions with infection, bone loss or both. One of them is the central bone grafting technique, which is a safe and effective treatment for nonunions of the tibia. The technique involves placement of autogenous cancellous bone from the iliac crest on the anterior surface of the interosseous membrane with the aim of creating a tibiofibular synostosis. We present the results of uncontrolled, retrospective and continuous series of ten patients treated by a central bone grafting technique for infected tibial nonunion with bone loss. Mean follow-up period was 12 (10-15) years. Most injuries were a result of war injuries. Clinically and radiologically confirmed bony healing with total consolidation of the graft was achieved in all patients within a period of 10-12 months without further bone grafting. The newly-formed bone mass was able to fulfil the mechanical and functional demands of everyday life activities. Once again, the central bone grafting technique has shown to be a safe, reliable and effective method of treatment for infected tibial nonunion with bone defect.     

Characterization of rat calvarial nonunion defects

This study examined the healing of nonunions by describing the histology and ultrastructural appearance of craniotomy defects as a model. Bone defects (3, 4 and 8 mm) were created in the calvaria of adult rats. Central and peripheral specimens of 8-mm defects were retrieved at 1, 3, 7, 10, 14, 21, 28 and 42 days and examined using both light and transmission electron microscopy. Specimens from the 3- and 4-mm defects were retrieved at 28 days and examined using light microscopy. In all sizes of defects, bony repair was consistently localized to the dural side of the defect. The 3- and 4 mm defects demonstrated the greatest degree of osseous bridging and evidence of normal osseous repair throughout the defect. The 8-mm defects repaired in general with the formation of nonunions which contained a small amount of bone at the periphery and fibrous connective tissue. Bone formation was evident at 10 days in the peripheral regions of the 8-mm defects and exhibited bony peninsulas with normal primary calcification fronts. Matrix vesicles containing hydroxyapatite-like crystals were present. In contrast, the central regions of the 8-mm defects were characterized by several islands of cartilage-like cells which stained metachromatically with toluidine blue. Transmission electron microscopy of this region at 14 days demonstrated a dense, collagenous extracellular matrix with matrix vesicles infiltrating the collagen bundles. There was no evidence of crystal formation in the matrix vesicles nor of calcification in the collagenous matrix. At 21 days, both the central and peripheral regions of the 8-mm calvarial nonunions were characterized by dense fibrous connective tissue repair and inactive fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biomechanical measurements on scaphoid bone screws in an experimental model

A number of screws commonly used for internal fixation in scaphoid bone fractures and nonunions are compared regarding biomechanical properties and clinical applicability. The experiments were carried out on models made of ash-wood, representing a reconstruction and fixation as is performed in a cortico-cancellous inlay bone graft for scaphoid non-union. For fixation use was made of 2.7 and 3.5 AO/ASIF cortical screws respectively, 4.0 AO/ASIF cancellous screws, Herbert screws, and a newly designed screw called the three components screw (D.K.S.). The models with implanted screws were tested for bending strength, tensile strength and torsion stability. No large differences between the various screws were found regarding the measured parameters, so that a small intra-osteal implant such as the Herbert screw and the D.K.S., which can be inserted easily and which gives a certain amount of interfragmentary compression, will be sufficient for osteosynthesis of the scaphoid bone. In case an intra-osteal implant is not available a single 3.5 AO/ASIF cortical screw, inserted following lag-screw principles, is recommended.     

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.     

Stiffness characteristics of the circular Ilizarov device as opposed to conventional external fixators

Ilizarov proposes the use of a special circular fixation device for treatment of bone defects and nonunions or for limb lengthening. Supposition was that the success of this method has to do with the specific mechanical behavior of the device. This behavior is a result of the configuration of the fixation elements. Therefore, a mechanical study of the sensitivity of the circular compression and distraction device (CDD) to configuration parameters was performed. The CDD was found to exhibit a nonlinear stiffness behavior, in particular under axial load. This may be favorable for the induction and tolerance of bone formation. Among the different parameters tested the ring radius was the most important with respect to stiffness. In general, the stiffness of the CDD allows adjustment during postoperative management. In magnitude, it is equal or lower when compared to other fixator types.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.     

Biologic adjuvants for fracture healing

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.     

Mesenchymal stem cell sheet transplantation combined with locally released simvastatin enhances bone formation in a rat tibia osteotomy model

Nonunion of fractured bones is a common clinical problem for orthopedic surgeons. This study aimed to investigate the effects of simvastatin locally applied from calcium sulfate (CS) combined with a mesenchymal stem cell (MSC) sheet on fracture healing. In vitro, the proliferation and differentiation of rat bone marrow–derived MSCs stimulated by simvastatin were investigated. In vivo, an osteotomy model was made in rat tibia, and fractured tibias were treated with CS, CS/simvastatin, CS/MSC sheet or simvastatin-loaded CS with MSC or untreated (control). Tibias were harvested at 2 or 8 weeks and underwent real-time quantitative polymerase chain reaction, x-ray, micro-CT and histological analysis. The expression levels of bone morphogenetic protein 2, alkaline phosphatase, osteocalcin, osteoprotegerin and vascular endothelial growth factor of simvastatin-induced MSCs increased with the concentrations of the simvastatin, significantly higher than those in the MSCs group. At 2 weeks, the CS/simvastatin/MSC sheet group showed significantly higher expressions of bone morphogenetic protein 2, alkaline phosphatase, osteocalcin, osteoprotegerin and vascular endothelial growth factor, with more callus formation around the fracture site compared with the other four groups. At 8 weeks, complete bone union was obtained in the CS/simvastatin/MSC sheet group. By contrast, newly regenerated bone tissue partially bridged the gap in the CS/simvastatin group and the CS/MSC sheet group; the control and CS group showed nonunion of the tibia. These results show that both simvastatin and the MSC sheet contributed to the formation of new bone and that the tibia fracture was completely healed by transplantation of the MSC sheet with locally applied simvastatin. Such MSC sheet with locally applied simvastatin might contribute to the treatment of fractures, bone delayed unions or nonunions in clinical practice.     

Magnitudes of local stress and strain along bony surfaces predict the course and type of fracture healing

A new quantitative tissue differentiation theory which relates the local tissue formation in a fracture gap to the local stress and strain is presented. Our hypothesis proposes that the amounts of strain and hydrostatic pressure along existing calcified surfaces in the fracture callus determine the differentiation of the callus tissue. The study compares the local strains and stresses in the callus as calculated from a finite element model with histological findings from an animal fracture model. The hypothesis predicts intramembranous bone formation for strains smaller approximately +/- 5% and hydrostatic pressures smaller than +/- 0.15 MPa. Endochondral ossification is associated with compressive pressures larger than about -0.15 MPa and strains smaller than +/- 15%. All other conditions seemed to lead to connective tissue or fibrous cartilage. The hypothesis enables a better understanding of the complex tissue differentiation seen in histological images and the mechanical conditions for healing delayed healing or nonunions.     

Lower extremity microsurgical reconstruction

LEARNING OBJECTIVES: After studying this article, the participant should be able to: 1. Understand the indications for the use of free-tissue transfer in lower extremity reconstruction. 2. Understand modalities to enhance the healing and care of soft tissue and bone before free-tissue transfer. 3. Understand the lower extremity reconstructive ladder and the place of free-tissue transfer on the ladder. 4. Understand the specific principles of leg, foot, and ankle reconstruction. 5. Understand the factors that influence the decision to perform an immediate versus a delayed reconstruction. Free-tissue transfer using microsurgical techniques is now routine for the salvage of traumatized lower extremities. Indications for microvascular tissue transplantation for lower extremity reconstruction include high-energy injuries, most middle and distal-third tibial wounds, radiation wounds, osteomyelitis, nonunions, and tumor reconstruction. The authors discuss the techniques and indications for lower extremity reconstruction.     

Effects of low-intensity AC and/or DC electromagnetic fields on cell attachment and induction of apoptosis

Rat tendon fibroblast (RTF) and rat bone marrow (RBM) osteoprogenitor cells were cultured and exposed to AC and/or DC magnetic fields in a triaxial Helmholtz coil in an incubator for up to 13 days. The AC fields were at 60 and 1000 Hz and up to 0.25 mT peak to peak, and the DC fields were up to 0.25 mT. At various combinations of field strengths and frequencies, AC and/or DC fields resulted in extensive detachment of preattached cells and prevented the normal attachment of cells not previously attached to substrates. In addition, the fields resulted in altered cell morphologies. When RTF and RBM cells were removed from the fields after several days of exposure, they partially reattached and assumed more normal morphologies. An additional set of experiments described in the Appendix corroborates these findings and also shows that low-frequency EMF also initiates apoptosis, i.e., programmed cell death, at the onset of cell detachment. Taken together, these results suggest that the electromagnetic fields result in significant alterations in cell metabolism and cytoskeleton structure. Further work is required to determine the relative effect of the electric and magnetic fields on these phenomena. The research has implications for understanding the role of fields in affecting bone healing in fracture nonunions, in cell detachment in cancer metastasis, and in the effect of EMF on organisms generally. Bioelectromagnetics 18:264–272, 1997. © Wiley-Liss, Inc.     

Recombinant human bone morphogenetic protein 2 and 7 inhibit the degeneration of intervertebral discs by blocking the Puma-dependent apoptotic signaling

Recombinant human bone morphogenetic proteins (rhBMPs) can stimulate bone formation and growth in the treatment of spinal fusions and nonunions. However, it is still unclear whether rhBMPs function in the prevention of intervertebral disc degeneration (IDD). Here, we discovered that BMP levels were decreased in IDD patients, which impaired the BMP/Smad (Mothers against decapentaplegic homologs) signaling. Conducting a microarray assay in Smad4-knockdown cells, we found that expression of PUMA (p53-upregulated modulator of apoptosis) was significantly induced. The molecular analysis revealed that Smad4 recruited HDAC1 (histone deacetylase 1) and the phosphorylated Smad1/5/8 to dock on the promoter of PUMA to repress its expression. The impairment of BMP/Smad signaling in IDD patients caused the significant induction of Puma-dependent apoptosis and resulted in the pathogenesis of IDD. In vitro knockdown of BMP receptors (BMPR1a and BMPR2) in nucleus pulposus (NP) cells could mimic the molecular changes of BMP/Smad signaling and Puma-dependent apoptotic signaling that were observed in IDD patients. Exposing NP cells to RITA (reactivating p53 and inducing tumor apoptosis) small molecule and rhBMP2 (or rhBMP7), we observed that rhBMP2/7 could significantly decrease protein levels of Puma and its downstream proapoptotic molecules, blocking cell apoptosis. Importantly, administration of rhBMPs in aged rats could inhibit the occurrence of IDD. Our results provide a link between BMP/Smad signaling and Puma-dependent apoptotic signaling, revealing a new mechanism of how BMPs contribute to IDD pathogenesis and providing evidence that rhBMPs may decrease apoptosis and improve the outcome of IDD.     

Outcome of diaphyseal forearm fracture-nonunions treated by autologous bone grafting and compression plating

Background

The treatment of forearm fracture-nonunions continues to represent a therapeutic challenge, and reported outcomes are moderate at best. Limiting aspects of this particular anatomic location include the relation between restoration of shaft length with the anatomy and long-term functional outcome of adjacent joints, as well as the risk of elbow and wrist stiffness related to prolonged immobilization. The present study was designed to assess the outcome of autologous bone grafting with compression plating and early functional rehabilitation in patients with forearm fracture non-unions.

Methods

Prospective follow-up study in 31 consecutive patients presenting with non-unions of the forearm diaphysis (radius, n = 11; ulna, n = 9; both bones, n = 11). Surgical revision was performed by restoring anatomic forearm length by autologous bone grafting of the resected non-union from the iliac crest and compression plating using a 3.5 mm dynamic compression plate (DCP) or limited-contact DCP (LC-DCP). The main outcome parameters consisted of radiographic bony union and functional outcome, as determined by the criteria defined by Harald Tscherne in 1978. Patients were routinely followed on a short term between 6 weeks to 6 months, with an average long-term follow-up of 3.6 years (range 2 to 6 years).

Results

Radiographically, a bony union was achieved in 30/31 patients within a mean time of 3.5 months of revision surgery (range 2 to 5 months). Clinically, 29/31 patients showed a good functional outcome, according to the Tscherne criteria, and 26/31 patients were able to resume their previous work. Two postoperative infections occurred, and one patient developed a persistent infected nonunion. No case of postoperative failure of fixation was seen in the entire cohort.

Conclusion

Revision osteosynthesis of forearm nonunions by autologous iliac crest bone grafting and compression plating represents a safe and efficacious modality for the treatment of these challenging conditions.     

Distraction osteogenesis of costochondral bone grafts in the mandible

Costochondral grafting for reconstruction of the Pruzansky type III mandible has given variable results. Lengthening of the rib graft by means of distraction had been advocated when subsequent growth of the grafted mandible is inadequate. This retrospective study reviews a series of patients with mandibular costochondral grafts who underwent subsequent distraction osteogenesis of the graft. A retrospective review identified two patient groups: group 1 consisted of individuals (n = 9) who underwent costochondral rib grafting of the mandible followed by distraction osteogenesis several months later at a rate of 1 mm/day. Group 2 consisted of patients with Pruzansky type II mandibles who had distraction osteogenesis without prior rib grafting (n = 9). The biomechanical parameters, orthodontic treatment regimens, and complications were examined versus patient age and quality of the rib graft. Distraction osteogenesis was successfully performed in six of the rib graft patients (group 1) and in all of the group 2 individuals. On the basis of the Haminishi scale, the computed tomographic scan appearance of the regenerate was classified as "standard or external" in six of the group 1 patients and as either "agenetic" or "pillar" (fibrous union) in the remaining three patients. In group 1, the average device was expanded 23 mm (range, 20 to 30 mm). Group 2 mandibular distraction results were all classified as either standard or external, and there was an average device expansion of 22.4 mm (range, 16 to 30 mm). The length of consolidation averaged 12.6 weeks in group 1, compared with 8.5 weeks in the traditional mandibular distraction patients (group 2). The mean shift of the dental midline to the contralateral side was 2.5 mm in group 1 versus 4.0 mm in group 2. Complex multiplanar and transport distractions were successfully performed on grafts of adequate bony volume. All four patients in group 1 with tracheostomies were successfully decannulated after consolidation. Rib graft distraction complications included pin tract infections in two patients, hardware failure with premature pin pullout in one patient, and evidence of fibrous nonunions in three young patients with single, diminutive rib grafts. In group 2, there were no distraction failures. Distraction osteogenesis can be successfully performed on costochondral rib grafts of the mandible; however, the complication rate is higher than in non-rib-graft patients. Performing the technique on older, more cooperative individuals seems to reduce this risk. In addition, placement of a double rib graft or an iliac bone graft of sufficient volume to create a neomandible with greater bone stock is an absolute requirement to decrease the risk of fibrous nonunion and provide a bone base of sufficient size for retention of the distraction device and manipulation of the regenerate.     

Antibody formation in mouse bone marrow. VIII. Dependence on potentially circulating memory cells: a study with parabiotic mice.

Mouse bone marrow barely contains antibody-producing plaque-forming cells (PFC) during the primary response to sheep red blood cells (SRBC). However, during the secondary response, the number of IgM, IgG, and IgA PFC in the bone marrow can rise to a level which surpasses the number of PFC in all the other lymphoid organs together. In the present paper we investigated whether the capacity of immune mice to react upon a booster injection of SRBC with a bone marrow PFC response can be transferred from immune to nonimmune mice. Therefore, mice primed with SRBC 6 months previously and nonprimed syngeneic mice were joined for parabiosis and were separated from each other at various intervals after joining. These separated mice were subsequently immunized with SRBC. It was found that, after 3 weeks of parabiosis, the nonprimed members reacted upon an injection of SRBC with a bone marrow IgM, IgG, and IgA PFC response as high as did the previously primed members. Furthermore it could be demonstrated by means of cell transfer experiments that, after a period of parabiosis of 3 weeks, the bone marrow and spleen of the normal mice contained about as many memory cells as the bone marrow and spleen of the immune mice. These results suggest that antibody formation in mouse bone marrow is dependent on a population of potentially circulating memory cells.     

Osteodensitometry in uncemented total hip arthroplasty using computertomography.

The aim of this investigation was to evaluate a new method developed for the measurement of bone mineral density and bone remodelling phenomena after total hip arthroplasty using computer tomography. Computertomography is a radiological technique to examine bone structures in high resolution. Using an extended scale it is possible to investigate bone scans and implants with fewer metal artifacts. For osteodensitometry measurement a special software (IMPact HIP) for the analysis of the data was used. The measured parameters were the overall bone mineral density (mg Calcium-Hydroxyapatite/ml) and the cortical bone structure. A standard scan mode enable to compare the computertomography scans at follow-up. Nineteen total hip arthroplasty patients (20 hips) with a mean age of 58 years (31-70) were operated on using an uncemented titanium alloy stem with a tapered design. The periprosthetic bone was assessed using computertomography-assisted osteodensitometry two weeks and one year after surgery. We observed a decrease of the overall bone mineral density (15%) and of the cortical bone structure (20%) one year after insertion of the stem in the proximal part of the femur. The area corresponds to the Gruen zones 1 and 7. On the other hand, a decrease of mineral density of 5% for the overall bone and of 3% for the cortical bone was found at the level of the tip of the stem, which corresponds to the Gruen zones 3, 4 and 5. Computertomography-assisted osteodensitometry allows to investigate the bone remodelling after total hip arthroplasty by separating the analysis of the overall bone mineral density and of the cortical structure. The present method is a reliable tool for quality-control in total hip arthroplasty.     

Composite tissue allografts in rats: IV. Graft-versus-host disease in recipients of vascularized bone marrow transplants.

This laboratory has used a composite tissue allograft model as a vehicle for studies on a new type of bone marrow transplant, the vascularized bone marrow transplant. The model consists of a rat hind limb transplant that incorporates integumentary musculoskeletal, and lymphopoietic tissues. These transplants, in comparison with conventional marrow transplants, have the advantage of providing a syngeneic microenvironment and immediate engraftment of both mature and progenitor hemopoietic cells at the time of transplantation. The characteristics of graft-versus-host disease were studied in this model. Lewis X Brown Norway F1 (LBN RT-1(1+n)) rats received hind limbs from Lewis (LEW RT-1(1)) donors (n = 19). Animals were observed daily for signs of graft-versus-host disease. Necropsies were performed. A minority of animals developed lethal disease (7 of 19 recipients) and demonstrated cachexia with concomitant histopathologic changes of the disease. Acute and chronic groups emerged with distinct clinical courses, which are similar to other models of this disease. Recipients of vascularized bone marrow transplants (limb transplants) showed clinical and histopathologic changes of the disease. The transplants may be used as a model of graft-versus-host disease in humans. Most interestingly, the transplant has a lower incidence of disease compared with other methods of bone marrow transplantation and represents an alternative to conventional bone marrow transplantation, which deserves further exploration. It may be possible to develop a new technique for bone marrow transplantation based on this surgical approach. It is proposed that the transfer of vascularized blocks of bone/marrow into prospective recipients as opposed to cellular bone marrow transplants may be preferable.     

Human bone marrow lymphocytes. III. Polyclonal activation of B lymphocytes in human bone marrow measured by a direct plaque-forming cell assay.

Lymphocytes from the bone marrow and peripheral blood of the same normal individuals were assayed simultaneously for blast transformation as well as polyclonal activation with differentiation to antibody-forming cells after stimulation with pokeweed mitogen. Blastogenic responses were measured by tritiated thymidine incorporation and antibody-forming cell assay. There was no significant difference between the blastogenic responses of lymphocytes in the peripheral blood compared to the bone marrow of the same individuals. However, differentiation to antibody-forming cells measured by the plaque-forming cell response was significantly greater in lymphocytes in the bone marrow as compared to peripheral blood of the same individuals. These studies demonstrate that the lymphocytes in human bone marrow are at a stage of differentiation whereby they can be readily induced to differentiation toward antibody production by polyclonal activation, even more so than peripheral blood lymphocytes. This supports the concept that the bone marrow is a major source of immunoglobulin production in man.