Calcite incorporated in silica/collagen xerogels mediates calcium release and enhances osteoblast proliferation and differentiation.

Multiphasic silica/collagen xerogels are biomaterials designed for bone regeneration. Biphasic silica/collagen xerogels (B30) and triphasic xerogels (B30H20 or B30CK20) additionally containing hydroxyapatite or calcite were demonstrated to exhibit several structural levels. On the first level, low fibrillar collagen serves as template for silica nanoparticle agglomerates. On second level, this silica-enriched matrix phase is fiber-reinforced by collagen fibrils. In case of hydroxyapatite incorporation in B30H20, resulting xerogels exhibit a hydroxyapatite-enriched phase consisting of hydroxyapatite particle agglomerates next to silica and low fibrillar collagen. Calcite in B30CK20 is incorporated as single non-agglomerated crystal into the silica/collagen matrix phase with embedded collagen fibrils. Both the structure of multiphasic xerogels and the manner of hydroxyapatite or calcite incorporation have an influence on the release of calcium from the xerogels. B30CK20 released a significantly higher amount of calcium into a calcium-free solution over a three-week period than B30H20. In calcium containing incubation media, all xerogels caused a decrease in calcium concentration as a result of their bioactivity, which was superimposed by the calcium release for B30CK20 and B30H20. Proliferation of human bone marrow stromal cells in direct contact to the materials was enhanced on B30CK20 compared to cells on both plain B30 and B30H20.

Bioinspired calcium phosphate mineralization on Net-Shape-Nonwoven chitosan scaffolds stimulates human bone marrow stromal cell differentiation.

Chitosan fibers were processed using the Net-Shape-Nonwoven (NSN) technique in order to create porous scaffolds which were functionalized in two bioinspired ways: collagen type I coating and unique mineralization with organically modified hydroxyapatite (ormoHAP). While collagen is common to enhance cell attachment on surfaces, the electric-field assisted migration and deposition of ormoHAP on the surface of the NSN-scaffolds is a novel technique which enables sub-micrometer sized mineralization while maintaining the original pore structure. Microscopy revealed fast attachment and morphological adaptation of the cells on both, the pure and the functionalized NSN-scaffolds. Remarkably, the cell number of osteogenically induced hBMSC on ormoHAP-modified NSN-scaffolds increased 3.5-5 fold compared to pure NSN-scaffolds. Osteogenic differentiation of hBMSC/osteoblasts was highest on collagen-functionalized NSN-scaffolds. RT-PCR studies revealed gene expression of ALP, BSP II, and osteocalcin to be high for all NSN-scaffolds. Overall, the NSN-scaffold functionalization with collagen and ormoHAP improved attachment, proliferation, and differentiation of hBMSC and therefore revealed the remarkable potential of their application for the tissue engineering of bone.

Manipulation of osteoclastogenesis: Bioactive multiphasic silica/collagen composites and their effects of surface and degradation products.

The intent of the present study was to demonstrate that multiphasic silica/collagen xerogels are able to manipulate cellular processes. These xerogels were prepared by a sol-gel approach allowing the incorporation of mineral phases. The resulting nanocomposites are designed as biomaterial for bone regeneration. Human osteoclasts derived from peripheral blood mononuclear cells were cultured both indirectly and directly, either in presence of different xerogel types or on their surface, to investigate the factor with the main influence on osteoclastogenesis. To this end, the incorporation of a third phase to silica/collagen xerogels was used to affect osteoclastogenesis. In cell culture, ambient ion conditions controlled by both the degradation products of the xerogel and the bioactivity-dependent ion release and reprecipitation were shown to have the main effect on osteoclast specific enzyme tartrate-resistant acid phosphatase (TRAP) 5b. Late stage of osteoclastogenesis characterized by resorption was strongly dependent on the xerogels composition. Surface chemistry of the xerogels was displayed to play an important role in osteoclast resorption. Biphasic silica/collagen xerogels and triphasic xerogels with calcium carbonate offered widespread resorbed areas, whereas hydroxyapatite containing xerogels showed distinctly reduced resorption. The incorporation of strontium carbonate and phosphate, respectively, as third phase changed TRAP 5b activity dose-dependently and inhibited resorption within 21 days. Quantitative evaluation on osteoclast differentiation was carried out using biochemical methods (TRAP 5b, cathepsin K) and was supported by confocal laser scanning microscopy and scanning electron microscopy (SEM). Qualitative estimation of resorption was carried out by SEM.

Electric field-assisted formation of organically modified hydroxyapatite (ormoHAP) spheres in carboxymethylated gelatin gels.

UNLABELLED: A biomimetic strategy was developed in order to prepare organically modified hydroxyapatite (ormoHAP) with spherical shape. The technical approach is based on electric field-assisted migration of calcium ions and phosphate ions into a hydrogel composed of carboxymethylated gelatin. The electric field as well as the carboxymethylation using glucuronic acid (GlcA) significantly accelerates the mineralization process, which makes the process feasible for lab scale production of ormoHAP spheres and probably beyond. A further process was developed for gentle separation of the ormoHAP spheres from the gelatin gel without compromising the morphology of the mineral. The term ormoHAP was chosen since morphological analyses using electron microscopy (SEM, TEM) and element analysis (EDX, FT-IR, XRD) confirmed that carboxymethylated gelatin molecules use to act as organic templates for the formation of nanocrystalline HAP. The hydroxyapatite (HAP) crystals self-organize to form hollow spheres with diameters ranging from 100 to 500nm. The combination of the biocompatible chemical composition and the unique structure of the nanocomposites is considered to be a useful basis for future applications in functionalized degradable biomaterials. STATEMENT OF SIGNIFICANCE: A novel bioinspired mineralization process was developed based on electric field-assisted migration of calcium and phosphate ions into biochemically carboxymethylated gelatin acting as organic template. Advantages over conventional hydroxyapatite include particle size distribution and homogeneity as well as achievable mechanical properties of relevant composites. Moreover, specifically developed calcium ion or phosphate ion release during degradation can be useful to adjust the fate of bone cells in order to manipulate remodeling processes. The hollow structure of the spheres can be useful for embedding drugs in the core, encapsulated by the highly mineralized outer shell. In this way, controlled drug release could be achieved, which enables advanced strategies for threating bone-related diseases, e.g. osteoporosis and multiple myeloma.

The impact of heat treatment on interactions of contact-poled biphasic calcium phosphates with proteins and cells.

A number of studies have reported improved bone integration for calcium phosphate based materials electrically "poled" by an external electric field prior to implantation. In our study we investigated the effects of electrical polarization of a biphasic ceramic composed of 80% hydroxyapatite and 20% ß-tricalcium phosphate. As contact poling involves elevated temperatures as a prerequisite for inducing charge, we used two reference types: samples without any heat treatment and poling, and samples with no poling but heat treatment identical to that of the poled samples. All heat-treated samples (poled or unpoled) showed an improved wettability, which was attributed to a reduced hydrocarbon contamination. Heat treatment alone provoked an accelerated spreading of osteoblast-like cells, whereas on poled samples a retarded cell spreading was observed. While proliferation and several differentiation markers were not influenced by either heat treatment or poling, the release of proinflammatory cytokines interleukin-6 and -8 was significantly reduced for all heat-treated samples, irrespective of additional electrical poling. The study demonstrated that the behaviour of cells in contact with poled biphasic ceramics was influenced by two parameters: heating and charge. Our data revealed that heating of the calcium phosphate ceramics had a much more pronounced effect on cell behaviour than charge.

Sulfated hyaluronan and chondroitin sulfate derivatives interact differently with human transforming growth factor-ß1 (TGF-ß1).

This study demonstrates that the modification of hyaluronan (hyaluronic acid; Hya) and chondroitin sulfate (CS) with sulfate groups leads to different binding affinities for recombinant human transforming growth factor-ß1 (TGF-ß1) for comparable average degrees of sulfation (DS). In general, Hya derivates showed higher binding strength than CS derivatives. In either case, a higher degree of sulfation leads to a stronger interaction. The high-sulfated hyaluronan sHya3 (average DS≈3) exhibited the tightest interaction with TGF-ß1, as determined by surface plasmon resonance and enzyme-linked immunosorbent assay. The binding strength was significantly weakened by carboxymethylation. Unmodified Hya and low-sulfated, native CS showed weak or no binding affinity. The interaction characteristics of the different sulfated glycosaminoglycans are promising for incorporation into bioengineered coatings of biomaterials to modulate growth factor binding in medical applications.

Effect of silica and hydroxyapatite mineralization on the mechanical properties and the biocompatibility of nanocomposite collagen scaffolds.

A recently established materials concept of biomimetic composites based on silica, collagen, and calcium phosphates was adapted for the preparation of porous scaffolds suitable for tissue engineering applications. Mineralization was achieved by directed nucleation of silica on the templating organic phase during a sol-gel process with or without addition of hydroxyapatite. Both mineral phases (25 wt %, individually or combined in equal shares) influenced the scaffold's morphology at the nanoscale. Enhancement of apparent density and compressive strength was similar for silica or hydroxyapatite mineralization; however the stiffening effect of hydroxyapatite was much higher. All scaffold modifications provided proper conditions for adhesion, proliferation, and osteogenic differentiation of human bone marrow stromal cells. The open porosity allowed cells to migrate throughout the scaffolds while maintaining their viability, both confirmed by MTT staining and confocal laser scanning microscopy. Initial cell distributions were graduated due to collagen mineralization, but balanced out over the cultivation time of 28 days. RT-PCR analyses revealed higher gene expression of ALP but lower expression of BSP II and osteocalcin because of collagen mineralization. The results demonstrate that both silica and hydroxyapatite offer comparable possibilities to tailor mechanical properties of collagen-based scaffolds without being detrimental to in vitro biocompatibility.

Mineralization at the interface of implants.

Osseointegration of implants is crucial for the long-term success of oral implants. Mineralization of the bone's extracellular matrix as the ultimate step of a mature bone formation is closely related to implant osseointegration. Osteogenesis at oral implants is a complex process, driven by cellular and acellular phenomena. The biological process of the maintenance and emergence of minerals in the vicinity of oral implants is influenced to a great extent by biophysical parameters. Implant-related structural and functional factors, as well as patient-specific factors, govern the features of osteogenesis. To understand the influence of these factors in peri-implant bone mineralization, it is important to consider the basic biological processes. Biological and crystallographic investigations have to be applied to evaluate mineralization at implant surfaces at the different hierarchical levels of analysis. This review gives insight into the complex theme of mineral formation around implants. Special focus is given to new developments in implant design and loading protocols aimed at accelerating osseointegration of dental implants.

Design and performance of a bioreactor system for mechanically promoted three-dimensional tissue engineering.

There is currently considerable interest in increasing the response of mesenchymal cells to physical forces, and numerous loading devices have been used to increase the formation of skeletal tissue in vivo and in vitro. We have developed a bioreactor system to apply cyclic strains on three-dimensional specimens over a range of 0-20,000 mustrain. The piezoelectric-driven mechanism allows the precise adjustment and control over load-related deformations of tissue, as shown by finite-element calculations of deformation of a collagen gel under load. We present the design of the bioreactor and its performance in specimens of tissue containing activated osteoblasts and chondrocytes. Biaxial tissue straining at 2,000 mustrain led to a substantial increase in the number of both types of cell compared with unstimulated controls. The synthesis of cell-specific extracellular matrix proteins increased when physiological loads (2,000 mustrain) were applied in the bioreactor, whereas higher deformations (20,000 mustrain) resulted in a reduction in proliferation and differentiation of cells. The mechanisms whereby mechanical stimulation leads to a defined cell reaction are not known, but the application of physiological micromovements in extracorporeal tissue chambers is a promising approach to the formation of hard tissue.

Principles of bone formation driven by biophysical forces in craniofacial surgery.

Biophysical forces, particularly mechanical loading and electromagnetic signals, are important regulators of bone formation. Indeed, the regenerative capacity of bony tissue is largely the result of the bone's capacity to recognise the functional environment required for the emergence and maintenance of a structurally intact bone. Biophysical methods of stimulation have therefore been introduced and have proved successful in clinical practice with craniofacial bones. Distraction osteogenesis, application of ultrasound, calculated transfer of stresses, and exposure to an electromagnetic field are some examples of biophysically driven approaches to influencing bone formation. The purpose of this review is to provide an insight into cellular and tissue models that are used to study the effects of biophysical stimuli on bone.

Basic reactions of osteoblasts on structured material surfaces.

In order to assess how bone substitute materials determine bone formation in vivo it is useful to understand the mechanisms of the material surface/tissue interaction on a cellular level. Artificial materials are used in two applications, as biomaterials alone or as a scaffold for osteoblasts in a tissue engineering approach. Recently, many efforts have been undertaken to improve bone regeneration by the use of structured material surfaces. In vitro studies of bone cell responses to artificial materials are the basic tool to determine these interactions. Surface properties of materials surfaces as well as biophysical constraints at the biomaterial surface are of major importance since these features will direct the cell responses. Studies on osteoblastlike cell reactivity towards materials will have to focus on the different steps of protein and cell reactions towards defined surface properties. The introduction of new techniques allows nowadays the fabrication of materials with ordered surface structures. This paper gives a review of present knowledge on the various stages of osteoblast reactions on material surfaces, focused on basic cell events under in vitro conditions. Special emphasis is given to cellular reactions towards ordered nano-sized topographies.

Aspects of collagen mineralization in hard tissue formation.

Collagen is the dominant fibrous protein not only in connective tissues but also in hard tissues, bone, dentin, cementum, and even the mineralizing cartilage of the epiphyseal growth plate. It comprises about 80-90% (by weight) of the organic substance in demineralized dentin and bone. When collagen fibers are arranged in parallel to form thicker bundles, as in lamellar bone and cementum, interior regions may be less mineralized; in dentin, however, the collagen fibers form a network and collagen fibers are densely filled with a mineral substance. In the biomineralization of collagen fibers in hard tissues, matrix vesicles play a fundamental role in the induction of crystal formation. The mineralization of matrix vesicles precedes the biomineralization of the collagen fibrils and the intervening ground substance. In addition, immobilized noncollagenous fibrous macromolecules, bound in a characteristic way to the fibrous collagen surface, initiate, more intensely than collagen, mineral nucleation in the hard tissue matrix.

[Osteoblast reaction on SLA and microgrooved implant surfaces]. / Primäre Osteoblastenreaktionen auf SLA- und mikrostrukturierten Implantatoberflächen.

The osseointegration process of dental implants depends on the tissue reaction at the tissue-implant interface. Osteoblasts are the main cells responsible for the regulation of osteoinduction. The manner and kinetics of the tissue reaction crucially depend on the interaction between osteoblasts and the morphology of the implant surface. The aim of this study was to investigate osteoblast behaviour on different implant surfaces (smooth, microgrooved, SLA) under standardized conditions. For this in vitro investigation we used primary bovine osteoblasts. Attachment kinetics, proliferation rate and synthesis of bone-associated proteins were used as parameters for cell reaction. The results demonstrate that both attachment and adhesion strength of the primary cell surface interaction was higher on the microgrooved surfaces than on SLA surfaces. The proliferation rate of cells and the synthesis of bone-specific proteins were higher on microgrooved surfaces in contrast to SLA surfaces. Ultrastructural analysis revealed phenotypic osteoblast-like cells on smooth and microgrooved surfaces, whereas cells on SLA surfaces showed a more fibroblastic appearance. This study demonstrates that the morphology of the implant surface determined the subsequent osteoblast reaction. An optimal cell reaction was found at surfaces which are smooth in the microenvironment of osteoblasts.

[Peri-implant bone formation around cylindrical and conical implant systems]. / Tierexperimentelle Evaluation des periimplantären Knochens bei zylindrischen gegenüber konischen Implantattypen.

BACKGROUND: The treatment of patients with early or immediately loaded dental implants has renewed interest in the behavior of osseointegration at the implant surface. Whereas it is generally accepted that peri-implant tissue formation and mineralization are dependent on the local mechanical environment in the interface zone, controversies exist concerning the impact of implant design on peri-implant bone formation. The aim of the present study was the in vivo evaluation of peri-implant bone formation by two different implant systems: cylindrical (ITI) versus conical (ILI). MATERIAL AND METHOD: A total of 60 implants (30 ITI and 30 ILI) were placed in the cranial and caudal part of the tibia of eight Göttinger minipigs. Half of the minipigs were sacrificed at 7 days and 28 days of osseointegration. Implant-containing bone specimens were prepared for histological and ultrastructural investigations. RESULTS: Histological and scanning electron-microscopic investigations showed a direct contact of bone-like minerals over the whole implant surface from day 7 of implant/bone interaction. Whereas the ILI implant showed direct contact up to the top of the crestal bone, ITI implants demonstrated a crestally located narrow gap without ossification over the whole experimental period. CONCLUSION: Our investigations support the hypothesis of an implant design-inherent emergence and maintenance of crestal bone.

Biological and biophysical principles in extracorporal bone tissue engineering. Part III.

Over the last decade extracorporal bone tissue engineering has moved from laboratory to clinical application. The restoration of maxillofacial bones from cell harvesting through product manufacture and end-use has benefited from innovations in the fields of biomechanical engineering, product marketing and transplant research. Cell/scaffold bone substitutes face a variety of unique clinical challenges which must be addressed. This overview summarises the recent state of the art and future anticipations in the transplantation of extracorporally fabricated bone tissues.

Biological and biophysical principles in extracorporal bone tissue engineering. Part II.

The aim of this review is to characterise the biological and biophysical background of in vitro bone tissue engineering. The paper focuses on basic principles in extracorporal engineering of bone-like tissues, considering parameters such as scaffold design, tissue construction, bioreactors, and cell stimulation in vivo and in vitro. Scaffolds have a key function concerning cellular invasion and bone formation. The intra-architectural scaffold geometry, as well as the scaffold material, play an important role in the process of bone regeneration. Various types of bioreactors have been tested for their utility in bone substitute fabrication that is clinically effective and reproducible. Sophisticated bioreactor systems are those that mimic the three-dimensional morphology and the mechanical situation of bones. Mechanical stimulation as well as other biophysical stimuli appear to be critical factors for proliferation and differentiation of bone cells and for bone mineral and structure formation. Furthermore an enhancement of bone regeneration by application of chemical stimulation factors is discussed.

Biological and biophysical principles in extracorporal bone tissue engineering. Part I.

Advances in the field of bone tissue engineering have encouraged physicians to introduce these techniques into clinical practice. Bone tissue engineering is the construction, repair or replacement of damaged or missing bone in humans or animals. Engineering of bone can take place within the animal body or extracorporal in a bioreactor for later grafting into the body. Appropriate cell types and non-living substrata are minimal requirements for an extracorporal tissue engineering approach. This review discusses the biological and biophysical background of in vitro bone tissue engineering. Biochemical and biophysical stimuli of cell growth and differentiation are regarded as potent tools to improve bone formation in vitro. The paper focuses on basic principles in extracorporal engineering of bone-like tissues, intended to be implanted in animal experiments and clinical studies. Particular attention is given in this part to the contributions of cell and material science to the development of bone-like tissues. Several approaches are at the level of clinical applicability and it can be expected that widespread use of engineered bone constructs will change the surgeon's work in the near future.

Ultrastructural characterization of the implant/bone interface of immediately loaded dental implants.

Primary stability and an optimized load transfer are assumed to account for an undisturbed osseointegration process of implants. Immediate loaded newly designed titanium dental implants inserted in the mandible of minipigs were used for the characterization of the interfacial area between the implant surface and the surrounding bone tissue during the early healing phase. Histological and electron microscopical studies were performed from implant containing bone specimens. Two different load regimens were applied to investigate the load related tissue reaction. Histological and electron microscopical analysis revealed a direct bone apposition on the implant surfaces, as well as the attachment of cells and matrix proteins in the early loading phase. A striking finding of the ultrastructural immunocytochemical investigations was the synthesis and deposition of bone related proteins (osteonectin, fibronectin, fibronectin receptor) by osteoblasts from day one of bone/biomaterial interaction. Calcium-phosphate needle-like crystallites were newly synthesized in a time-related manner directly at the titanium surface. No difference in the ultrastructural appearance of the interface was found between the two loading groups. Our experimental data suggest that loading of specially designed implants can be performed immediately after insertion without disturbing the biological osseointegration process.

Evaluation of accuracy of insertion of dental implants and prosthetic treatment by computer-aided navigation in minipigs.

The survival of loaded implants is critically dependent on their biomechanical stability. We have used a computer-guided navigation technique to evaluate the accuracy of computer-assisted insertion for immediately-loaded implants in minipigs. On the basis of computed tomographical data, the Robodent system was used for preoperative planning and guidance of inserting the implant. An optical tracking system allowed positioning of the implant and immediate prosthetic rehabilitation by inserting it in a plaster model and during the operation. Postoperative computed tomograms (CT) showed that the implants were placed precisely in the preoperatively planned position. The accuracy achieved corresponded well with the spatial resolution of the CT used. Immediate placement of the prefabricated crowns resulted in favourable occlusal positioning. Histological cross-sections showed that the implants were biomechanically stable. The accuracy of insertion of oral implants illustrated here suggests that insertion and prosthetic modelling of implants may benefit from computer-assisted navigation.

[Evaluating angiogenesis and osteogenesis modified by vascular endothelial growth factor (VEGF)]. / Beurteilung der Angiogenese und Osteogenese unter dem Einfluss von Vascular endothelial growth factor (VEGF).

PURPOSE: This intention of this study was to investigate the influence of controlled release of vascular endothelial growth factor (VEGF) on angiogenesis and osteogenesis in a mandibular defect model. METHODS: A total of 56 rabbits were operated and bicortical holes were placed in the mandible. The defects were filled with collagen type I implants, collagen implants complexed with 0.8-microgram VEGF165, or left without any filling. After 3, 7, 14, and 28 days specimens were taken and histologic, histomorphometric, and immunohistologic analyses were carried out concerning density of vessels, total surface of vessels, bone surface, and bone density. RESULTS: The number of vessels was increased in all groups up to 14 days, followed by physiologic regression in the control groups, whereas the study group showed persistently high numbers. The density of regenerated bone was significantly higher in the study group. CONCLUSION: The activation of angiogenesis using VEGF165 leads to more intensive angiogenesis and bone regeneration.