Molecular, cellular and pharmaceutical aspects of synthetic hydroxyapatite bone substitutes for oral and maxillofacial grafting

Werner Götz and Spyridon N. Papageorgiou

Current Pharmaceutical Biotechnology 18(1): 95-106, 2017.

Abstract: Bone grafts are widely used for augmentation procedures in oral and maxillofacial surgery, with autogenous bone being the gold standard. Recently, the focus of research has shifted towards synthetic bone substitutes, as no second surgery is needed and large quantities of graft can easily be provided. Within the broad range of bone substitutes, synthetic hydroxyapatite has drawn much attention, as they are considered to be biocompatible, non-immunogenic, osteoconductive and osteoinductive. Scope of this review is to summarize existing knowledge concerning the molecular, cellular and pharmaceutical aspects of synthetic bone substitutes for oral and maxillofacial grafting.

Nanostructured synthetic bone replacement material for the treatment of bone defects

B. Kienast, H. Neumann, F. Brüning-Wolter, R. Wendlandt, R. Kasch, A. P. Schulz

Trauma und Berufskrankheit 18(4): 308-318, 2016.

Abstract: Autologous bone grafting is still the gold standard for the reconstruction of bone defects from fractures. Complication rates up to 30% have been described in the literature for bone grafting procedures. Thus, research is concerned with the development of alternative bone substitutes, which satisfy the high requirements of bone inductivity, bone conductivity and osteogenesis. This study compared the complications and fracture healing rates of patients with bone defects treated with synthetic silicatesubstituted calcium phosphate. Concerning complications and fracture healing rates, the results of our study are comparable to the results of autologous bone grafting in the available literature.

Despite limited comparability, the complication rate of the treatment of bone defects with silicatesubstituted nanohydroxyapatite seems lower than the combined complication rates for bone harvesting and grafting together. In our study synthetic bone substitutes did not show poorer results concerning complication rates and fracture healing compared to the gold standard. These materials should be considered as an alternative for the treatment of bone defects in fractures of the extremities and pelvis. The strength of the study lies in the prospective and consecutive design. A computed tomography of all included patients would have been desirable but could not be performed due to financial and radiation hygiene reasons.

Comparison of bone substitutes in a tibia defect model in Wistar-rats

Ganz C, Xu W, Holzhüter G, Götz W, Vollmar B, Gerber T

Key Engineering Materials Vols. 493-494: 732-738, 2012.

Abstract: Various bone graft substitutes were used in clinical practice in the treatment of bone defects after trauma or osteoporosis. Many synthetic biomaterials were developed in recent years primarily based on hydroxyapatite (HA). NanoBone® is a nanocrystalline hydroxyapatite (HA) embedded in a porous matrix of silica (SiO2). The ratio of HA:SiO2 varied between 76:24 (wt%; NanoBone) and 61:39 (wt%; Nanobone® S). The two bone substitutes NB and NB S and a natural bovine bone substitute Bio-Oss® (BO) were evaluated by means of implantation in the tibia of the rat. The aim of this study was to analyze the remodeling process and to measure new bone formation and degradation after implantation of these biomaterials. A tibia defect model was used for all investigations with testing periods of 12, 21 and 84 days. (n=5 for each time point).

The results showed, that all bone grafts were well accepted by the host tissue without inflammatory reactions. In comparison to the biomaterial BO, NanoBone and NanoBone S were quickly degraded, whereas autologous proteins were incorporated into nanopores. New bone formation was statistically higher in NanoBone S compared to Bio-Oss in defect area after 84 days implantation. The presence of osteoclasts in tissue sections were demonstrated by TRAP- and ED1- immunohistology.
(NOTE: NanoBone S (HA:SiO2 61:39 wt%) is commercially available in the US.)

Osteogenic capacity of nanocrystalline bone cement in a weight-bearing defect at the ovine tibial metaphysis.

Harms, et. al.

Int Journal of Nanomedicine 7:2883-2889, 2012.

Abstract: The synthetic material Nanobone® (hydroxyapatite nanocrystallites embedded in a porous silica gel matrix) was examined in vivo using a standardized bone defect model in the ovine tibial metaphysis. A standardized 6x12x24-mm bone defect was created below the articular surface of the medial tibia condyles on both hind legs of 18 adult sheep. The defect on the right side was filled with Nanobone, while the defect on the contralateral side was left empty. The tibial heads of six sheep were analyzed after 6, 12, and 26 weeks each. The histological and radiological analysis of the defect on the control side did not reveal any bone formation after the total of 26 weeks. In contrast, the microcomputed tomography analysis of the defect filled with Nanobone showed a 55%, 72%, and 74% volume fraction of structures with bone density after 6, 12, and 26 weeks, respectively. Quantitative histomorphological analysis after 6 and 12 weeks revealed an osteoneogenesis of 22%, and 36%, respectively. Hematoxylin and eosin sections demonstrated multinucleated giant cells on the surface of the biomaterial and resorption lacunae, indicating osteoclastic resorptive activity. Nanobone appears to be a highly potent bone substitute material with osteoconductive properties in a loaded large animal defect model, supporting the potential use of Nanobone also in humans.

Lateral augmentation of the mandible in minipigs with a synthetic nanostructured hydroxyapatite block.

Kirchhoff, et. al.

J Biomed Mater Res B 96:342-250, 2011.

Abstract: The purpose of this study was to evaluate biomaterial degradation and new bone formation after implantation of a nanostructured hydroxyapatite (HA) grafting block. Furthermore, physical characteristics of the biomaterial were measured. The biomaterial consists of nanostructured HA embedded in a porous matrix of silica (SiO2) gel. The blocks with two different contents of silica (group A: 24 wt % and group B: 39 wt %) were fixed with titanium screws at the lateral aspect of the mandible of minipigs (n = 5). The specific surface areas of both blocks were measured using Brunauer– Emmett–Teller (BET) equation and mercury intrusion. In all animals, the wound healing was uneventful. After 5 weeks, the biomaterial percentage was 51.5% ± 12.1% for group A and 33.2% ± 5.9% for group B (p = 0.017). New bone formation accounted to 7.6% ± 6.0% for group A and 15.3% ± 8.3% for group B (p = 0.126) after 5 weeks. After 10 weeks, further resorption of the biomaterial led to percentages of 30.6% ± 10.0% for group A and 12.1% ± 6.7% for group B (p = 0.000). After 10 weeks, new bone formations were measured to be 34.1% ± 10.8% in group A and 39.9% ± 13.5% in group B (p = 0.383). The rate of degradation of the biomaterial is controlled by the composition of the material. A higher content of silica gel matrix leads to faster degradation of the biomaterial. The formation of new bone failed to show a significant difference between both groups. (NOTE: NanoBone S (HA:SiO2 61:39 wt%) is commercially available in the US.)

Evaluation of injectable silica-embedded nanohydroxyapatite bone substitute in a rat tibia defect model

Xu W, Ganz C, Weber U, Adam M, Holzhüter G, Wolter D, Frerich B, Vollmar B, Gerber T

Int J Nanomedicine. 6:1543-52, 2011.

Abstract: In clinical practice, vertebral compression fractures occur after trauma and osteoporosis. Kyphoplasty is a minimally invasive procedure using bone filler material for the treatment of such fractures. A full synthetic injectable bone substitute (SIBS) was manufactured by means of spray drying. The aim of this study was to characterize the SIBS and to analyze the remodeling process during degradation of the biomaterial and new bone formation after implantation. SIBS is an aqueous suspension of donut-like microparticles. These microparticles consist of nanocrystallites of synthetic hydroxyapatite embedded in amorphous silica gel. After implantation of SIBS in a proximal tibial diaphyseal defect in 52 rats, grafts were harvested for subsequent analysis on different days. Newly formed bone originating from endosteum was observed on day 6. Hematomas in the medullary space and cortical wounds disappeared on day 12. The wound region was completely replaced by a composite of newly formed cancellous bone, extracellular matrix, and SIBS. At day 63 the cortical defect was fully healed by bone, while newly formed bone in the medullary space almost disappeared and was replaced with bone marrow. In conclusion, SIBS demonstrated a unique structure with osteoinductive and bioresorbable properties, which induced fast bone regeneration. Therefore, a clinical application of SIBS for kyphoplasty is promising.

In vivo analysis of biocompatibility and vascularization of the synthetic bone grafting substitute NanoBone®.

Abshagen, et. al.

J Biomed Mater Res A 91:557-566, 2009

Abstract: One of the major challenges in the application of bone substitutes is adequate vascularization and biocompatibility of the implant. Thus, the temporal course of neovascularization and the microvascular inflammatory response of implants of NanoBone® (fully synthetic nanocrystalline bone grafting material) were studied in vivo by using the mouse dorsal skinfold chamber model. Angiogenesis, microhemodynamics, and leukocyte-endothelial cell interaction were analyzed repetitively after implantation in the center and in the border zone of the implant up to 15 days. Both NanoBone granules and plates exhibited high biocompatibility comparable to that of cancellous bone, as indicated by a lack of venular leukocyte activation after implantation. In both synthetic NanoBone groups, signs of angiogenesis could be observed even at day 5 after implantation, whereas granules showed higher functional vessel density compared with NanoBone plates. The angiogenic response of the cancellous bone was markedly accelerated in the center of the implant tissue. Histologically, implant tissue showed an ingrowth of vascularized fibrous tissue into the material combined with an increased number of foreign body giant cells. In conclusion, NanoBone, particularly in granular form, showed high biocompatibility and high angiogenic response, thus improving the healing of bone defects. Our results underline that, beside the composition and nanostructure, the macrostructure is also of importance for the incorporation of the biomaterial by the host tissue.

Early matrix change of a nanostructured bone grafting substitute in the rat.

Xu W, Holzhüter G, Sorg H, Wolter D, Lenz S, Gerber T, Vollmar B

J Biomed Mater Res B Appl Biomater. 91(2): 692-9, 2009.

Abstract: A nanocrystalline bone substitute embedded in a highly porous silica gel matrix (NanoBone®) has previously been shown to bridge bone defects by an organic matrix. As the initial host response on the bone graft substitute might be a determinant for subsequent bone formation, our present purpose was to characterize the early tissue reaction on this biomaterial. After implantation of 80 mg of NanoBone into the adipose neck tissue of a total of 35 rats, grafts were harvested for subsequent analysis at days 3, 6, 9, 12, and 21. The biomaterial was found encapsulated by granulation tissue which partly penetrated the implant at day 3 and completely pervaded the graft at day 12 on implantation. Histology revealed tartrate-resistant acid phosphatase (TRAP)-positive giant cells covering the biomaterial. ED1 (CD68) immunopositivity of these cells further indicated their osteoclast-like phenotype. Scanning electron microscopy revealed organic tissue components within the periphery of the graft already at day 9, whereas the central hematoma region still presented the silica-surface of the biomaterial. Energy dispersive X-ray spectroscopy further demonstrated that the silica gel was degraded faster in the peripheral granulation tissue than in the central hematoma and was replaced by organic host components by day 12. In conclusion, the silica gel matrix is rapidly replaced by carbohydrate macromolecules. This might represent a key step in the process of graft degradation on its way toward induction of bone formation. The unique composition and structure of this nanoscaled biomaterial seem to support its degradation by host osteoclast-like giant cells.

Immunohistochemical characterization of nanocrystalline hydroxyapatite silica gel (NanoBone®) osteogenesis: A study on biopsies from human jaws.

Götz, et. al.

Clin Oral Res 19:1016-1026, 2008.

Abstract: Bone substitute biomaterials may be osteogenic, osteoconductive or osteoinductive. To test for these probable characteristics in a new nanoporous grafting material consisting of nanocrystalline hydroxyapatite embedded in a porous silica gel matrix (NanoBone ), applied in humans, we studied biopsies from 12 patients before dental implantation following various orofacial augmentation techniques with healing times of between 3.5 and 12 months. Sections from decalcified specimens were investigated using histology, histochemistry [periodic acid Schiff, alcian blue staining and tartrate-resistant acid phosphatase (TRAP)] and immunohistochemistry, with markers for osteogenesis, bone remodeling, resorption and vessel walls (alkaline phosphatase, bone morphogenetic protein-2, collagen type I, ED1, osteocalcin, osteopontin, runx2 and Von-Willebrand factor). Histologically, four specific stages of graft transformation into lamellar bone could be characterized. During early stages of healing, bone matrix proteins were absorbed by NanoBone granules, forming a proteinaceous matrix, which was invaded by small vessels and cells. We assume that the deposition of these molecules promotes early osteogenesis in and around NanoBone and supports the concomitant degradation probably by osteoclast-like cells. TRAP-positive osteoclast-like cells were localized directly on the granular surfaces. Runx2- immunoreactive pre-osteoblasts, which are probably involved in direct osteogenesis forming woven bone that is later transformed into lamellar bone, were attracted. Graft resorption and bone apposition around the graft granules appear concomitantly. We postulate that NanoBone has osteoconductive and biomimetic properties and is integrated into the host’s physiological bone turnover at a very early stage.

Nanostructuring of Biomaterials - a pathway to bone grafting substitute.

Gerber, et. al.

European Journal of Trauma 32:132-140, 2006.

Abstract: The bone substitute NanoBone® consists of nanocrystalline hydroxyapatite embedded in a highly porous matrix of silica gel. It promotes the healing of bone defects and is degraded by osteoclasts during bone remodeling. The present study investigates the interactions of NanoBone® with bone tissue. Granules of NanoBone® were implanted in defects of critical size in the mandible of minipigs. Samples were taken after 5 and 10 weeks and demineralized. The composition of the implanted granules was analyzed by means of transmission and scanning electron microscopy and EDX. Enzyme- and immunohistochemistry was used to investigate organic components of NanoBone® granules that arised after implantation in the host. EDX demonstrated that 5 weeks after implantation the silica gel was degraded and replaced by an organic matrix. Ultrastructurally, the matrix appeared amorphous with only single collagen fibrillae. PAS-staining indicated the presence of carbohydrates. Immunohistochemically, the bone proteins osteopontin, osteocalcin and BMP-2 were found as constituents of the new matrix. Alkaline phosphatase activity was located in osteoblasts and newly formed bone on NanoBone® and focally in particles. Osteoclasts with ruffled borders, sealing zones, and acid phosphatase activity were situated in resorption lacunae at granule surfaces not covered by new bone. In vivo, the silica gel of NanoBone® is replaced by bone matrix glycoproteins with known functions in attraction, adhesion, and differentiation
 of bone cells as osteoblasts and osteoclasts. We assume that the deposition of these molecules supports the early phase of NanoBone® degradation by osteoclasts and promotes the production of new bone tissue.