Two Distinctive BMP-Carriers Induce Zonal Chondrogenesis and Membranous Ossification,Respectively; Geometrical Factors of Matrices for Cell-Differentiation

A partially purified BMP preparation was combined with a fibrous glass membrane (FGM) or porous particles of hydroxyapatite (PPHAP), and then implanted subcutaneously into the backs of rats. As a control of these new carriers, a conventional carrier of insoluble bone matrix (IBM) was also used. These new geometrically different solid-state carriers induced tissues in quite different manners. FGM/BMP implants induced cartilage formation within the entire inner area of the membrane accompanied by a small amount of bone formation on the surface of the membrane. In contrast, PPHAP/ BMP implants induced only bone within the pores of PPHAP without any detectable cartilage formation. Enzyme-linked immunosorbent assay revealed that the type II collagen content in FGM/BMP was six times higher than that in IBM/BMP, while there was no detectable type II collagen in PPHAP/BMP. The results were explained by the geometric properties of the two distinctive carriers.     

Carrier dependent cell differentiation of bone morphogenetic protein-2 induced osteogenesis and chondrogenesis during the early implantation stage in rats

To evaluate the osteoinductive effects of recombinant human bone morphogenetic protein (rhBMP)-2 during the early stages of rat ectopic bone formation, we prepared two distinct carriers. Two carriers, insoluble bone matrix (IBM) and fibrous glass membrane (FGM) were combined with rhBMP-2 and implanted into the backs of rats to evaluate the osteoinductive effects of the two rhBMP-2 carrier systems. Insoluble bone matrix particle size was 320 to 620 microm. Fibrous glass membrane was constructed from unwoven glass fibers 1 microm in diameter. Alkaline phosphatase (ALP) activity and type II collagen were detected in IBM/rhBMP-2 at 5 days postimplantation. Calcium (Ca) was also detected in IBM/rhBMP-2 at 7 and 9 days postimplantation. In contrast, ALP and type II collagen were detected in FGM/rhBMP-2 at 7 days. Calcium was undetected, indicating that the bone formation in IBM/rhBMP-2 proceeded faster than in FGM/rhBMP-2 during the early stage of BMP-induced osteogenesis. In addition, mRNA expression level of KDR, a receptor for vascular endothelial growth factor, was also increased in IBM/rhBMP-2. To investigate the in vivo release profile of rhBMP-2, iodine 125 ((125)I)-labeled BMP-2-incorporating IBM and FGM implants were inserted into the back subcutis of mice. More than 60% of the rhBMP-2 was released from the IBM/rhBMP-2 carrier within 1 day after implantation, whereas 50% of the rhBMP-2 was released from the FGM/rhBMP-2 10 days postimplantation. These results indicated that osteo- and chondrogenesis depends highly upon the geometry of the carrier and the in situ retention of rhBMP-2 during the early stage of rhBMP-2 induced bone formation.     

Ectopic osteoinduction and early degradation of recombinant human bone morphogenetic protein-2-loaded porous beta-tricalcium phosphate in mice

The present study investigated the ectopic osteoinduction and early degradation of recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded porous beta-tricalcium phosphate (beta-TCP) in mice. The porous beta-TCP with 50 microg of rhBMP-2 (n = 25) and porous beta-TCP (control group, n = 25) were implanted into muscle pouches in the right and left thigh of 28-day-old mice (n = 25), respectively. At every time point (3, 7, 14, 21 and 28 days after implantation), five mice were euthanized and the histological examinations of implantation sites were performed. In addition, the alkaline phosphatase (ALP) activity was also quantitatively analyzed. For the rhBMP-2-loaded group, blood vessel formation and immature cartilage was observed within the porous beta-TCP 3 days after implantation. Mature cartilage was observed 7 days after implantation of rhBMP-2-loaded porous beta-TCP. Newly formed woven bone, lamellar bone as well as marrow were observed 14 and 21 days after implantation of the rhBMP-2-loaded porous beta-TCP. Lamellar bone and marrow were observed 28 days after implantation of the rhBMP-2-loaded porous beta-TCP. For the control group, no bone or cartilage was observed at all time points. However, multinucleated giant cells and fibrous tissues were observed in the control group at 7 and 28 days after implantation, respectively. At 21 and 28 days after implantation, porous beta-TCP was observed to fragment indicating early degradation of the porous beta-TCP in both groups. In addition, ALP was observed to be significantly higher in the rhBMP-2-loaded beta-TCP as compared to the control beta-TCP. It was concluded from this study that the rhBMP-2-loaded porous beta-TCP induced blood vessel and ectopic bone formation.     

Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants inserted at an ectopic site in rabbits

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is known for its osteoinductive potential in bone tissue engineering. Calcium phosphate (Ca-P) cements are injectable, osteoconductive ceramic materials in which a macroporous structure can be induced during the setting reaction. In this study, the osteoinductive capability of rhBMP-2 loaded porous Ca-P cement was evaluated. Porous Ca-P cement discs were made and loaded with rhBMP-2 in vitro and implanted subcutaneously in the back of New Zealand white rabbits. The implantation period was either 2 or 10 weeks. Histological analysis of retrieved specimens revealed evident bone formation in the rhBMP-2 loaded Ca-P cement discs (pore fill: 18+/-6%) after 10 weeks of implantation. Bone formation occurred only in rhBMP-2 loaded porous Ca-P cement discs. Degradation of the Ca-P cement could not be confirmed after 10 weeks of implantation. The scaffold maintained its shape and stability during this time period. We conclude that porous Ca-P cement is a suitable carrier material for ectopic bone engineering.     

Bone formation in CaP-coated and noncoated titanium fiber mesh

The osteogenic activity of calcium phosphate (CaP)-coated and noncoated porous titanium (Ti) fiber mesh loaded with cultured syngeneic osteogenic cells after prolonged in situ culturing was compared in a syngeneic rat ectopic assay model. Rat bone marrow (RBM) cells were loaded onto the CaP-coated and noncoated Ti scaffolds using either a droplet or a suspension loading method. After loading, the RBM cells were cultured for 8 days in vitro. Thereafter, implants were subcutaneously placed in 39 syngeneic rats. The rats were euthanized and the implants retrieved at 2, 4, and 8 weeks postoperatively. Further, in the 8 week group fluorochrome bone markers were injected at 2, 4, and 6 weeks. Histological analysis demonstrated that only the CaP-coated meshes supported bone formation. The amount of newly formed bone varied between single and multiple spheres to filling a significant part of the mesh porosity. In the newly formed bone, osteocytes embedded in a mineralized matrix could be observed clearly. On the other hand, in the noncoated titanium implants, abundant deposition of calcium-containing material was seen. This deposit lacked a bonelike tissue organization. Further analysis revealed that the cell-loading method did not influence the final amount of bone formation. In CaP-coated implants the accumulation sequence of the fluorochrome markers showed that bone formation started on the mesh fibers. In conclusion, our results prove that the combination of a thin CaP coating, Ti-mesh, and RBM cells can indeed generate ectopic bone formation after prolonged in vitro culturing. No effect of the loading method was observed on the final amount of bone.     

Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits

In this study, the osteoinductive properties of porous calcium phosphate (Ca-P) cement loaded with bone morphogenetic protein 2 (rhBMP-2) were evaluated and compared with rhBMP-2 loaded absorbable collagen sponge (ACS). Discs with a diameter of 8mm were loaded with a buffer solution with or without 10 microg rhBMP-2 and inserted in 8mm full thickness cranial defects in rabbits for 2 and 10 weeks of implantation. Histological analysis revealed excellent osteoconductive properties of the Ca-P material. It maintained its shape and stability during the implantation time better than the ACS but showed no degradation like the ACS. Quantification of the Ca-P cement implants showed that bone formation was increased significantly by administration of rhBMP-2 (10 weeks pore fill: 53.0+/-5.4%), and also reached a reasonable amount without rhBMP-2 (43.1+/-10.4%). Remarkably, callus-like bone formation outside the implant was observed frequently in the 2 weeks rhBMP-2 loaded Ca-P cement implants, suggesting a correlation with the presence of growth factor in the surrounding tissue. However, an additional in vitro assay revealed an accumulative release of no more than 9.7+/-0.9% after 4 weeks. We conclude that: (1). Porous Ca-P cement is an appropriate candidate scaffold material for bone engineering. (2). Bone formation can be enhanced by lyophilization of rhBMP-2 on the cement. (3). Degradation of porous Ca-P cement is species-, implantation site- and implant dimension-specific.     

Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation

Three different types of porous hydroxyapatite with pore sizes of 100-200 micrometer in diameter-porous particles of hydroxyapatite (PPHAP), porous blocks of hydroxyapatite (PBHAP), and honeycomb-shaped hydroxyapatite (HCHAP)-were compared in terms of their abilities to induce osteogenesis when implanted subcutaneously with recombinant human BMP-2 into rats and extracted at 1, 2, 3, and 4 weeks. Histologically, direct bone formation occurred in PPHAP and PBHAP while only endochondral ossification took place in HCHAP. Interestingly, cartilage in the central zones and bone in the orifice zones of the tunnels of the HCHAP were observed at 2 weeks. After 3 weeks, the cartilage disappeared and bone formation occurred throughout the inner surface of the tunnels of the HCHAP, always leaving space for capillaries within the tunnels. Alkaline phosphatase activity and osteocalcin content were the highest in HCHAP among the three hydroxyapatite implants. These results clearly indicate that BMP-induced bone formation is highly dependent on the geometry of the carrier, which provides feasible structural factors for vascularization.     

Geometry of artificial ECM: sizes of pores controlling phenotype expression in BMP-induced osteogenesis and chondrogenesis

This study aims to elucidate the feasible geometry of the scaffolds in bone and periodontal tissue engineering. Several biomaterials with different geometries are compared in terms of their patterns of ectopic BMP-induced chondrogenesis and osteogenesis. The materials include a honeycomb-shaped hydroxyapatite (HCHAP) with different tunnel sizes, a laser-perforated collagen membrane (LPM), and CPSA bioglass fibers. Implanted pellets were removed at 1-4 weeks and analyzed for bone and cartilage formation histologically and biochemically. Porous particles of hydroxyapatite (PPHAP), porous blocks of hydroxyapatite (PBHAP), and LPM did not induce detectable cartilage formation. In straight tunnel structures with various diameters in honeycomb-shaped hydroxyapatite (HCHAP), tunnels with smaller diameters (approximately 0.1 mm) induced cartilage followed by bone formation, while one with a larger diameter (0.35 mm) directly induced bone formation within the tunnels. It is concluded that the "vasculature-inducing geometry" of the carrier as an ECM is crucially important for osteogenesis.     

Modulation of stromal cell-derived factor-1/CXC chemokine receptor 4 axis enhances rhBMP-2-induced ectopic bone formation

Enhancement of in vivo mobilization and homing of endogenous mesenchymal stem cells (MSCs) to an injury site is an innovative strategy for improvement of bone tissue engineering and repair. The present study was designed to determine whether mobilization by AMD3100 and/or local homing by delivery of stromal cell-derived factor-1 (SDF-1) enhances recombinant human bone morphogenetic protein-2 (rhBMP-2) induced ectopic bone formation in an established rat model. Rats received an injection of either saline or AMD3100 treatment 1 h before harvesting of bone marrow for in vitro colony-forming unit-fibroblasts (CFU-F) culture or the in vivo subcutaneous implantation of absorbable collagen sponges (ACSs) loaded with saline, recombinant human bone morphogenetic protein-2 (rhBMP-2), SDF-1, or the combination of SDF-1 and rhBMP-2. AMD3100 treatment resulted in a significant decrease in CFU-F number, compared with saline, which confirmed that a single systemic AMD3100 treatment rapidly mobilized MSCs from the bone marrow. At 28 and 56 days, bone formation in the explanted ACS was assessed by microcomputed tomography (μCT) and histology. At 28 days, AMD3100 and/or SDF-1 had no statistically significant effect on bone volume (BV) or bone mineral content (BMC), but histology revealed more active bone formation with treatment of AMD3100, loading of SDF-1, or the combination of both AMD3100 and SDF-1, compared with saline-treated rhBMP-2 loaded ACS. At 56 days, the addition of AMD3100 treatment, loading of SDF-1, or the combination of both resulted in a statistically significant stimulatory effect on BV and BMC, compared with the saline-treated rhBMP-2 loaded ACS. Histology of the 56-day ACS were consistent with the μCT analysis, exhibiting more mature and mineralized bone formation with AMD3100 treatment, SDF-1 loading, or the combination of both, compared with the saline-treated rhBMP-2 loaded ACS. The present study is the first that provides evidence of the efficacy of AMD3100 and SDF-1 treatment to stimulate trafficking of MSCs to an ectopic implant site, in order to ultimately enhance rhBMP-2 induced long-term bone formation.     

Bone formation in calcium-phosphate-coated titanium mesh

The osteogenic activity of porous titanium fiber mesh and calcium phosphate (Ca-P)-coated titanium fiber mesh loaded with cultured syngeneic osteogenic cells was compared in a syngeneic rat ectopic assay model. In 30 syngeneic rats, (Ca-P)-coated and non-coated porous titanium implants were subcutaneously placed either without or loaded with cultured rat bone marrow (RBM) cells. Fluorochrome bone markers were injected at 2, 4, and 6 weeks. The rats were sacrificed, and the implants were retrieved at 2, 4, and 8 weeks post-operatively. Histological analysis demonstrated that none of the (Ca-P)-coated and non-coated meshes alone supported bone formation at any time period. In RBM-loaded implants, bone formation started at 2 weeks. At 4 weeks, bone formation increased. However, at 8 weeks bone formation was absent in the non-coated titanium implants, while it had remained in the (Ca-P)-coated titanium implants. Also, in (Ca-P)-coated implants more bone was formed than in non-coated samples. In general, osteogenesis was characterized by the occurrence of multiple spheres in the porosity of the mesh. The accumulation sequence of the fluorochrome markers showed that the newly formed bone was deposited in a centrifugal manner starting at the center of a pore. Our results show that the combination of Ti-mesh with RBM cells can indeed generate bone formation. Further, our results confirm that a thin Ca-P coating can have a beneficial effect on the bone-generating properties of a scaffold material.     

Repair of long intercalated rib defects in dogs using recombinant human bone morphogenetic protein-2 delivered by a synthetic polymer and beta-tricalcium phosphate

Long intercalated defects in canine ribs can be repaired successfully using porous beta-tricalcium phosphate (beta-TCP) cylinders, infused with a biodegradable polymer (poly D,L-lactic acid-polyethylene block copolymer) containing recombinant human bone morphogenetic protein-2 (rhBMP-2). We previously reported the successful regeneration of bony rib and periosteum defects using beta-TCP cylinders containing 400 microg of rhBMP-2. To reduce the amount of rhBMP-2 and decrease the time required for defect repair, we utilized a biodegradable polymer carrier, in combination with rhBMP-2 and the porous beta-TCP cylinders. An 8 cm long section of rib bone was removed and replaced with an implant comprised of the porous beta-TCP cylinders and the polymer containing 80 microg of rhBMP-2. Six weeks after surgical placement of the beta-TCP cylinder/polymer/BMP-2 implants, new rib bone with an anatomical configuration and mechanical strength similar to the original bone was regenerated at the defect site. The stiffness of the regenerated ribs at 3, 6, and 12 weeks after implantation of the composite implant was significantly higher than that of ribs regenerated by implantation of rhBMP-2/beta-TCP implants. Thus, addition of the synthetic polymer to the drug delivery system for BMP potentiated the bone-regenerating ability of the implant and enabled the formation of mechanically competent rib bone. This new method appears to be applicable to the repair of intercalated long bone defects often encountered in clinical practice.     

β-TCP降解和rhBMP-2诱导成骨的关系

探讨β-TCP的降解率是否可以影响rhBMP-2的诱导成骨量.将不同降解率的β-TCP1和β-TCP2分别与rhBMP-2复合制备成不同降解率的两种复合物,β-TCP1/rhBMP-2和β-TCP2/rhBMP-2.然后分别种植于小鼠股部肌袋中.种植后分别进行组织学检查、组织形态计量、碱性磷酸酶和降解量测定.两种复合物种植后均有软骨和骨形成,成骨量随时间推移而增加.但是,种植后8周时β-TCP1/rhBMP-2组成骨量显著高于β-TCP2/rhBMP-2组(P＜0.05).种植后4周以前两组的ALP水平均随时间推移而升高(P＜0.05),但是,在4～8周间则无明显差异.此外,种植后8周时β-TCP1/rhBMP-2的降解量显著高于β-TCP2/rhBMP-2(P＜0.05).β-TCP的降解率可以影响BMP的诱导成骨量.     

Geometry of Artificial ECM: Sizes of Pores Controlling Phenotype Expression in BMP-Induced Osteogenesis and Chondrogenesis

This study aims to elucidate the feasible geometry of the scaffolds in bone and periodontal tissue engineering. Several biomaterials with different geometries are compared in terms of their patterns of ectopic BMP-induced chondrogenesis and osteogenesis. The materials include a honeycomb-shaped hydroxyapatite (HCHAP) with different tunnel sizes, a laser-perforated collagen membrane (LPM), and CPSA bioglass fibers. Implanted pellets were removed at 1-4 weeks and analyzed for bone and cartilage formation histologically and biochemically. Porous particles of hydroxyapatite (PPHAP), porous blocks of hydroxyapatite (PBHAP), and LPM did not induce detectable cartilage formation. In straight tunnel structures with various diameters in honeycomb-shaped hydroxyapatite (HCHAP), tunnels with smaller diameters (approximately 0.1 mm) induced cartilage followed by bone formation, while one with a larger diameter (0.35 mm) directly induced bone formation within the tunnels. It is concluded that the "vasculature-inducing geometry" of the carrier as an ECM is crucially important for osteogenesis.     

重组人骨形态发生蛋白2/骨修复材料的制备与性能

BACKGROUND: It has become a hotspot to prepare the bone repair material that exhibits natural bone structure and is used in combination with biological factors. OBJECTIVE: To prepare the recombinant human bone morphogenetic protein-2 (rhBMP-2)/bone repair material, and to evaluate its capacities of release, activity and ectopic osteoinduction. METHODS: A collagen-binding domain was added to the N-terminal of native rhBMP-2 that allowed bind to collagens in the bone repair material. Then, rhBMP-2/bone repair material was obtained through freeze-dried method. The releasing ability of rhBMP-2 in vitro was assayed by ELISA. C2C12 cell lines were loaded to the composite material with 0.25, 0.5 and 1 µg rhBMP-2, respectively. Afterwards, alkaline phosphatase activity was detected at 72 hours. The composite materials with 0, 2, 5 and 10 µg rhBMP-2 were implanted into the quadriceps of Sprague-Dawley rats, respectively. Alkaline phosphatase activity and the newly formed bone were detected at 2 and 4 weeks after implantation. The CY-7-labeled composite material was implanted into the quadriceps of Sprague-Dawley rats to observe its stability. RESULTS AND CONCLUSION: Substantially no rhBMP-2 from the rhBMP-2/bone repair material was released within 45 days. The alkaline phosphatase activity of C2C12 was in a rise with the increased concentration of rhBMP-2. The stability of the composite material in vivo was better, the alkaline phosphatase activity and ectopic bone formation increased as the concentration of rhBMP-2 rose. To conclude, the rhBMP-2/bone repair material preserves the stability of rhBMP-2, and improves ectopic osteoinduction ability.     

The effect of pretreating morselized allograft bone with rhBMP-2 and/or pamidronate on the fixation of porous Ti and HA-coated implants.

BMPs stimulate new bone formation, but may also accelerate bone resorption. We added rhBMP-2 and pamidronate to morselized bone allograft packed around uncemented HA-coated and non-coated porous Ti implants in sixteen dogs. Each dog received four implants where the allograft was added (1) nothing, (2) BMP, (3) BP, and (4) BMP+BP. After four weeks, the untreated control implants had better mechanical fixation than all other treatment groups. The rhBMP-2-treated group had abundant formation of new bone on and around the implant. However, almost all allografts were resorbed, rendering the implant mechanically unstable. In the pamidronate-treated group the allograft was preserved, but the implants were covered by fibrous tissue and there was almost no new bone formation. This was also the case for the combined BMP+BP group, although fibrous tissue was absent on these implants. The HA-coated implants had more than twice as good mechanical fixation and improved osseointegration compared to the corresponding Ti implants. RhBMP-2 raised the total metabolic turnover of bone within the allograft with a net negative result on implant fixation. Pamidronate virtually blocked bone metabolism, also when combined with rhBMP-2. The results warrant a conservative approach and emphasize the importance of identifying a therapeutic window for these substances prior to clinical use.     

A new bone-inducing biodegradable porous beta-tricalcium phosphate

A new type of degradable biomaterial with bone-inducing capacity was made by combining porous beta-tricalcium phosphate (beta-TCP) with a delivery system for recombinant human bone morphogenetic protein-2 (rhBMP-2). The BMP delivery system consisted of a block copolymer composed of poly-D,L-lactic acid with random insertion of p-dioxanone and polyethylene glycol (PLA-DX-PEG), a known biocompatible and biodegradable material. The efficacy of this biomaterial in terms of its bone-inducing capacity was examined by ectopic bone formation in the dorsal muscles of the mouse. In the beta-TCP implants coated with the PLA-DX-PEG polymer containing more than 0.0025% (w/w) of rhBMP-2, new ectopic bone tissues with marrow were consistently found on the surface of implants. The radiographic density of beta-TCP was diminished in a time-dependent manner. On histological examination, numerous multinucleated osteoclasts with positive tartrate-resistant acid-phosphatase (TRAP) staining were noted on the surface of the beta-TCP. These experimental results indicate that beta-TCP implants coated with synthetic rhBMP-2 delivery system might provide effective artificial bone-graft substitutes with osteoinductive capacity and biodegradable properties. In addition, this type of biomaterial may require less rhBMP-2 to induce significant new bone mass.     

Collagen-infiltrated porous hydroxyapatite coating and its osteogenic properties: in vitro and in vivo study

Bone-implant interface is critical for the early fixation of orthopedic implants. In this study, porous hydroxyapatite (HA) coatings were prepared through a liquid precursor plasma spraying process and were infiltrated with the collagen, alone and with the additional incorporation of recombinant human bone morphogenetic protein-2 (rhBMP-2) and RGD peptide (RGD). The results showed significantly improved mesenchymal stem cell (MSC) adhesion, proliferation, and differentiation on collagen-modified HA coatings, partially benefited from the formation of a fibrous network due to the self-reconstitution of collagen on the HA surface. Further enhancements on MSC proliferation and differentiation were generally observed through the additional incorporation of bone morphogenetic protein (BMP) and RGD. The osteoinductive and osteoconductive properties of the collagen/BMP-modified HA coatings were studied in vivo. Clear ectopic bone formation and significantly accelerated bone growth rate (29% increase, p < 0.05) have been observed after 1-month implantation of HA-collagen/rhBMP-2-coated Ti alloy samples into the rabbit muscle and dog femora, respectively. Overall, our results suggest that collagen-modified HA coating surface is a far superior substrate for cell attachment, proliferation, and differentiation, and collagen can be used an efficient carrier for BMP in vivo. Therefore, modification of HA coating with collagen is a simple but effective biomimetic approach to enhancing the osteointegration and early fixation of bone-implant interface.     

Preparation of beta-tricalcium phosphate microsphere-hyaluronic acid-based powder gel composite as a carrier for rhBMP-2 injection and evaluation using long bone segmental defect model

The specific objective of this study was to evaluate whether rhBMP-2-loaded bio-scaffolds can be used as effective rhBMP-2 carriers in the implantation of bone defect sites or poor bone quality in host bone. The rhBMP-2 release pattern test showed slow results in both groups, and a 1:9 ratio composition with a high water-absorption rate was selected for in vivo study. All animals euthanized after 9 weeks. The new bone formation and bone quantity and quality of fibular samples were examined. The results showed that the rhBMP-2 powder gel composite improved the new bone formation in the cortical bone and the marrow space. The length of new bone formation ratio of the rhBMP-2 loaded composite group was significantly higher than the powder gel group. The composite of powder gel seems to be a nice carrier, and slow release of rhBMP-2 can promote new bone formation in a segmental cortical bone defect after implantation.     

Bone-like tissue formation using an equine COLLOSS E-filled titanium scaffolding material

COLLOSS, a bovine extracellular matrix product containing native BMPs has already shown osteoinductive properties. To overcome problems with risk of transmissable spongiform encephalopathy (TSE) infection, an equine derived version was investigated in this study, named COLLOSS E. Disc- and tube-shaped implants were made from titanium fibre mesh. The central space of tubes was filled and the discs were impregnated with the COLLOSS E material to assess osteo-induction. These implants and non-loaded controls were implanted subcutaneously into the back of Wistar rats. After implantation periods of 2, 8, and 12 weeks, the implants were retrieved and sections were made. Histology showed a thin fibrous capsule surrounding the titanium mesh and a very mild tissue reaction. The disc implants, loaded or non-loaded, showed no bone formation at all. After 2 weeks of implantation 3 out of 5 of the loaded tubes showed bone formation with a mean of 0.3 mm2 areas of new formed bone, after 8 weeks 3 out of 6 and 0.7 mm2, and after 12 weeks this increased to 6 out of 6 and 1.0 mm2. In the non-loaded tubes only connective tissue in growth was seen. In conclusion, it was demonstrated that COLLOSS E material, loaded in a titanium fibre mesh tube shows osteoinductive properties. The effect of COLLOSS E has to be investigated further in orthotopic sites, which resemble more the final clinical application for bone reconstruction.     

Mineral apposition rates provide significant information on long-term effects in BMP-induced bone regeneration

In this study, a CaP biomaterial was used as a carrier for rhBMP-2. Biomaterials were investigated in calvarial and femoral defects using a rabbit animal model, with unloaded biomaterials serving as control. Fluorochrome labels were administered at days 14 and 70. Specimens were retrieved after 12 weeks for histological analysis. When area fractions were assessed by conventional histomorphometry, no significant effect of rhBMP-2 on the amounts of regenerated bone and residual biomaterial were seen by 12 weeks. After mineral appositional rate (MAR) measurement using double labels, calculation yielded significantly higher MARs for defects at both implantation sites, when compared with surrounding bone, whether or not biomaterials were loaded with rhBMP-2. Analyzing the effect of rhBMP-2, both defect sites showed significantly higher MARs in the rhBMP-2 group. MARs of bone surrounding the defects had also been elevated significantly by rhBMP-2 at calvarial and femoral implantation sites. It is concluded that MAR measurement is suitable to identify long-term effects of rhBMP-2 on bone formation at a time when conventional histomorphometry using fractional area determination is inadequate. Also, by MAR assessment, effects of rhBMP-2 on surrounding bone can be documented.