Louise Renton

Osteoinductive hydroxyapatite-coated titanium implants.

Previous studies have shown that heterotopic induction of bone formation by calcium phosphate-based macroporous constructs is set into motion by the geometry of the implanted substrata, i.e. a sequence of repetitive concavities assembled within the macroporous spaces. The aim of this study was to construct osteoinductive titanium implants that per se, and without the exogenous application of the osteogenic soluble molecular signals of the transforming growth factor-β supergene family, would initiate the induction of bone formation. To generate intrinsically osteoinductive titanium implants for translation in clinical contexts, titanium grade Ti-6A1-4V cylinders of 15 mm in length and 3.85 mm in diameter, with or without concavities, were plasma sprayed with crystalline hydroxyapatite resulting in a uniform layer of 30 μm in thickness. Before coating, experimental titanium implants were prepared with a sequence of 36 repetitive concavities 1600 μm in diameter and 800 μm in depth, spaced a distance of 1000 μm apart. Mandibular molars and premolars were extracted to prepare edentulous mandibular ridges for later implantation. Planar and geometric hydroxyapatite-coated titanium constructs were implanted in the left and right edentulized hemi-mandibles, respectively, after a healing period of 7-8 months, 3 per hemi-mandible. Three planar and three geometric implants were implanted in the left and right tibiae, respectively; additionally, planar and geometric constructs were also inserted in the rectus abdominis muscle. Six animals were euthanized at 30 and 90 days after implantation; one animal had to be euthanized 5 days after surgery and the remaining animal was euthanized 31 months after implantation. Undecalcified longitudinal sections were precision-sawed, ground and polished to 40-60 μm; all sections were stained with a modified Goldners trichrome. Undecalcified specimen block preparation was performed using the EXAKT precision cutting and grinding system. Histomorphometric analyses of bone in contact (BIC) showed that on day 30 there was no difference between the geometric vs. planar control implants; on day 90, the ratio of BIC to surface within the geometric implants was greater than on the standard planar implants in both mandibular and tibial sites; 31 months after implantation, selected concavities cut into the geometric implants harvested from the rectus abdominis muscle showed the spontaneous induction of bone formation with mineralized bone surfaced by osteoid seams. These data in non-human primates indicate that geometrically-constructed plasma-sprayed titanium implants are per se osteogenic, the concavities providing a unique microenvironment to initiate bone differentiation by induction.

The induction of bone formation by smart biphasic hydroxyapatite tricalcium phosphate biomimetic matrices in the non-human primate Papio ursinus

Long‐term studies in the non‐human primate Chacma baboon Papio ursinus were set to investigate the induction of bone formation by biphasic hydroxyapatite/p‐tricalcium phosphate (HA/β‐TCP) biomimetic matrices. HA/β‐TCP biomimetic matrices in a pre‐sinter ratio (wt%) of 40/60 and 20/80, respectively, were sintered and implanted in the rectus abdominis and in calvarial defects of four adult baboons. The post‐sinter phase content ratios were 19/81 and 4/96, respectively. Morphological analyses on day 90 and 365 showed significant induction of bone formation within concavities of the biomimetic matrices with substantial bone formation by induction and resorption/dissolution of the implanted matrices. One year after implantation in calvarial defects, 4/96 biphasic biomimetic constructs showed prominent induction of bone formation with significant dissolution of the implanted scaffolds. The implanted smart biomimetic matrices induce de novo bone formation even in the absence of exogenously applied osteogenic proteins of the transforming growth factor‐β(TGF‐β) superfamily. The induction of bone formation biomimetizes the remodelling cycle of the cortico‐cancellous bone of primates whereby resorption lacunae, pits and concavities cut by osteoclastogenesis are regulators of bone formation by induction. The concavities assembled in HA/β‐TCP biomimetic bioceramics are endowed with multifunctional pleiotropic self‐assembly capacities initiating and promoting angiogenesis and bone formation by induction. Resident mesenchymal cells differentiate into osteoblastic cell lines expressing, secreting and embedding osteogenic soluble molecular signals of the TGF‐β superfamily within the concavities of the biomimetic matrices initiating bone formation as a secondary response.

Synergistic induction of bone formation by hOP-1, hTGF-β3 and inhibition by zoledronate in macroporous coral-derived hydroxyapatites

Thirty coral-derived calcium carbonate-based macroporous constructs with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) were implanted in the rectus abdominis of three adult non-human primate Papio ursinus to investigate the intrinsic induction of bone formation. Macroporous constructs with 125 microg human recombinant osteogenic protein-1 (hOP-1) or 125 microg human recombinant transforming growth factor-beta(3) (hTGF-beta(3)) were also implanted. The potential synergistic interaction between morphogens was tested by implanting binary applications of hOP-1 and hTGF-beta(3) 5:1 by weight, respectively. To evaluate the role of osteoclastic activity on the implanted macroporous surfaces, coral-derived constructs were pre-loaded with 0.24 mg of bisphosphonate zoledronate (Zometa). To correlate the morphology of tissue induction with osteogenic gene expression and activation, harvested specimens on day 90 were analyzed for changes in OP-1 and TGF-beta(3) mRNA synthesis by quantitative real-time polymerase chain reaction (qRT-PCR). The induction of bone formation in 7% HA/CC solo correlated with OP-1 expression. Massive bone induction formed by binary applications of the recombinant morphogens. Single applications of hOP-1 and hTGF-beta(3) also resulted in substantial bone formation, not comparable however to synergistic binary applications. Zoledronate-treated macroporous constructs showed limited bone formation and in two specimens bone formation was altogether absent; qRT-PCR showed a prominent reduction of OP-1 gene expression whilst TGF-beta(3) expression was far greater than OP-1. The lack of bone formation by zoledronate-treated specimens indicates that osteoclastic activity on the implanted coral-derived constructs is critical for the spontaneous induction of bone formation. Indirectly, zoledronate-treated samples showing lack of OP-1 gene expression and absent or very limited bone formation by induction confirm that the spontaneous induction of bone formation by coral-derived macroporous constructs is initiated by secreted BMPs/OPs, in context the OP-1 isoform.

The induction of endochondral bone formation by transforming growth factor-β3: experimental studies in the non-human primate Papio ursinus

Transforming growth factor‐β3 (TGF‐β3), a multi‐functional growth modulator of embryonic development, tissue repair and morphogenesis, immunoregulation, fibrosis, angiogenesis and carcinogenesis, is the third mammalian isoform of the TGF‐β subfamily of proteins. The pleiotropism of the signalling proteins of the TGF‐β superfamily, including the TGF‐β proteins per se, are highlighted by the apparent redundancy of soluble molecular signals initiating de novo endochondral bone induction in the primate only. In the heterotopic bioassay for bone induction in the subcutaneous site of rodents, the TGF‐β3 isoform does not initiate endochondral bone formation. Strikingly and in marked contrast to the rodent bioassay, recombinant human (h)TGF‐β3, when implanted in the rectus abdominis muscle of adult non‐human primates Papio ursinus at doses of 5, 25 and 125 μg per 100 mg of insoluble collagenous matrix as carrier, induces rapid endochondral bone formation resulting in large corticalized ossicles by day 30 and 90. In the same animals, the delivery of identical or higher doses of theTGF‐β3 protein results in minimal repair of calvarial defects on day 30 with limited bone regeneration across the pericranial aspect of the defects on day 90. Partial restoration of the bone induction cascade by the hTGF‐β3 protein is obtained by mixing the hTGF‐β3 device with minced fragments of autogenous rectus abdominis muscle thus adding responding stem cells for further bone induction by the hTGF‐β3 protein. The observed limited bone induction in hTGF‐β3/treated and untreated calvarial defects in Papio ursinus and therefore by extension to Homo sapiens, is due to the influence of Smad‐6 and Smad‐7 down‐stream antagonists of the TGF‐β signalling pathway. RT‐PCR, Western and Northern blot analyses of tissue specimens generated by the TGF‐β3 isoform demonstrate robust expression of Smad‐6 and Smad‐7 in orthotopic calvarial sites with limited expression in heterotopic rectus abdominis sites. Smad‐6 and ‐7 overexpression in hTGF‐β3/treated and untreated calvarial defects may be due to the vascular endothelial tissue of the arachnoids expressing signalling proteins modulating the expression of the inhibitory Smads in pre‐osteoblastic and osteoblastic calvarial cell lines controlling the induction of bone in the primate calvarium.

TISSUE ENGINEERING: TGF-BETA SUPERFAMILY MEMBERS AND DELIVERY SYSTEMS IN BONE REGENERATION

The induction of bone formation requires three parameters that interact in a highly regulated process: soluble osteoinductive signals, capable responding cells, and a supporting matrix substratum or insoluble signal. The use of recombinant and naturally derived bone morphogenetic proteins and transforming growth factor beta(s) (TGF-beta(s)) has increased our understanding of the functions of these morphogens during the induction of endochondral bone formation. In addition, growing understanding of the cellular interactions of living tissues with synthetic biomaterials has led to the in vivo induction of bone formation using porous biomimetic matrices as an alternative to the use of autografts for bone regeneration. This review outlines the basis of bone tissue engineering by members of the TGF-beta superfamily, focusing on their delivery systems and the intrinsic induction of bone formation by specific biomimetic matrices with a defined geometry.

Soluble Signals and Insoluble Substrata

The repair and regeneration of bone is a complex process that is temporally and spatially regulated by soluble and insoluble signals (1). The initiation of bone formation during embryonic development and postnatal osteogenesis involves a complex cascade of molecular and morphogenetic processes that ultimately lead to the architectural sculpturing of precisely organized multicellular structures.

Biomimetic Smart Hydroxyapatite/Biphasic Tricalcium Phosphate Biomatrices Induce Bone Formation

Long-term studies in the non-human primate Papio ursinus were set to investigate the induction of bone formation in biphasic hydroxyapatite tricalcium phosphate (HA/TCP) biomimetic matrices, 20/80 and 40/60, respectively. Biomimetic matrices were implanted in the rectus abdominis and in calvarial defects of 4 adult Papio ursinus. Morphological analyses on day 90 and 365 showed significant induction of bone formation within concavities of the biomimetic matrices implanted in both heterotopic and orthotopic sites with resorption of the implanted biomimetic matrices. The smart biomimetic matrices induced de novo bone formation even in the absence of exogenously applied osteogenic proteins of the transforming growth factor-β superfamily.

Gene Expression and Bone Induction Regulated by Bioceramic Substratum

Bioceramic structures based on hydroxyapatite have been reported whic h are apable of inducing bone growth when implanted in muscle tissue and in the absence of exogenously applied growth factors. This spontaneous inductive response, which is extremely discriminating with regard to a number of material and host requirements, is triggered when the surface binds autologous osteoinductive BMPs such as BMP-7 and BMP-3 which then initiate a bone induction cascade. In this paper the response is reported for biphasic degradable substrata of hydroxyapatite – 33 % tricalcium phosphate ceramic, implanted in a soft tissue primate odel and where the physical distribution of co-existing phases has been manipulated. A remarkable a spect is the fact that significant and critical differences are observed in the express ion of mRNA markers of bone formation according to the composition and microstructure of the bioceram ic surface. The design of the ceramic substratum is shown to dramatically influence and reg ulat gene expression, driving the emergence of the osteogenic phenotype and the morphogenesis of bone as a secondary response. Introduction Bioceramic structures of hydroxyapatite have been reported which are capable of inducing bone growth when implanted in muscle tissue and in the absence of exogenously applied growth factors [1-3]. This spontaneous inductive response is somewhat elusive in that it is extremely discriminating with regard to a number of material and host require ments such as the animal model, the chemical composition, the presence of concavities of relatively large size and spherical contour, significant surface microporosity and area, even the physical size of implants. When the set of requirements are suitably met, however, the response appears to be quit e predictable and reproducible. Even though it is complex and not fully understood, the response is known to be triggered when the surface binds autologous osteoinductive BMPs such as BMP-7 and BMP-3 under suitable conditions, which then initiate a bone induction cascade. While hydroxyapatite (HAp) has proved to be particularly effective in eliciting this r esponse, it does present a disadvantage in bone applications in that it is considered essentially on-resorbable. Hence the rendering of composite materials containing HAp in a resorbable or de gradable form is relevant. One particular material, the so-called biphasic calcium phosphate ( BCP) ceramic, is of particular interest in this regard. The conventional BCP material comprises a fine distribution of TCP in HAp matrix, usually with TCP:HAp ratio in the range 25:75 to 40:60 [4]. The material develops a resorption front in vivo through solution and also osteoclastic resorption, whic h is much more Key Engineering Materials Online: 2003-05-15 ISSN: 1662-9795, Vols. 240-242, pp 639-642 doi:10.4028/www.scientific.net/KEM.240-242.639