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Triply periodic minimal surfaces (TPMSs) are found to be promising microarchitectures for bone substitutes owing to their low weight and superior mechanical characteristics. However, existing studies on their application are incomplete because they focus solely on biomechanical or in vitro aspects. Hardly any in vivo studies where different TPMS microarchitectures are compared have been reported. Therefore, we produced hydroxyapatite-based scaffolds with three types of TPMS microarchitectures, namely Diamond, Gyroid, and Primitive, and compared them with an established Lattice microarchitecture by mechanical testing, 3D-cell culture, and in vivo implantation. Common to all four microarchitectures was the minimal constriction of a sphere of 0.8 mm in diameter, which earlier was found superior in Lattice microarchitectures. Scanning by μCT revealed the precision and reproducibility of our printing method. The mechanical analysis showed significantly higher compression strength for Gyroid and Diamond samples compared with Primitive and Lattice. After in vitro culture with human bone marrow stromal cells in control or osteogenic medium, no differences between these microarchitectures were observed. However, from the TPMS microarchitectures, Diamond- and Gyroid-based scaffolds showed the highest bone ingrowth and bone-to-implant contact in vivo . Therefore, Diamond and Gyroid designs appear to be the most promising TPMS-type microarchitectures for scaffolds produced for bone tissue engineering and regenerative medicine. Impact Statement Extensive bone defects require the application of bone grafts. To match the existing requirements, scaffolds based on triply periodic minimal surface (TPMS)-based microarchitectures could be used as bone substitutes. This work is dedicated to the investigation of mechanical and osteoconductive properties of TPMS-based scaffolds to determine the influencing factors on differences in their behavior and choose the most promising design to be used in bone tissue engineering.

The early phase of bone healing is a complex and poorly understood process. With additive manufacturing, we can generate a specific and customizable library of bone substitutes to explore this phase. In this study, we produced tricalcium phosphate-based scaffolds with microarchitectures composed of filaments of 0.50 mm in diameter, named Fil050G, and 1.25 mm named Fil125G, respectively. The implants were removed after only 10 days in vivo followed by RNA sequencing (RNAseq) and histological analysis. RNAseq results revealed upregulation of adaptive immune response, regulation of cell adhesion, and cell migration-related genes in both of our two constructs. However, significant overexpression of genes linked to angiogenesis, regulation of cell differentiation, ossification, and bone development was observed solely in Fil050G scaffolds. Moreover, quantitative immunohistochemistry of structures positive for laminin revealed a significantly higher number of blood vessels in Fil050G samples. Furthermore, µCT detected a higher amount of mineralized tissue in Fil050G samples suggesting a superior osteoconductive potential. Hence, different filament diameters and distances in bone substitutes significantly influence angiogenesis and regulation of cell differentiation involved in the early phase of bone regeneration, which precedes osteoconductivity and bony bridging seen in later phases and as consequence, impacts the overall clinical outcome.

Additive manufacturing is a key technology required to realize the production of a personalized bone substitute that exactly meets a patient's need and fills a patient-specific bone defect. Additive manufacturing can optimize the inner architecture of the scaffold for osteoconduction, allowing fast and reliable defect bridging by promoting rapid growth of new bone tissue into the scaffold. The role of scaffold microporosity/nanoarchitecture in osteoconduction remains elusive. To elucidate this relationship, we produced lithography-based osteoconductive scaffolds from tricalcium phosphate (TCP) with identical macro- and microarchitecture, but varied their nanoarchitecture/microporosity by ranging maximum sintering temperatures from 1000 °C to 1200 °C. After characterization of the different scaffolds' microporosity, compression strength, and nanoarchitecture, we performed in vivo studies that showed that ingrowth of bone as an indicator of osteoconduction significantly decreased with decreasing microporosity. Moreover, at the 1200 °C peak sinter temperature and lowest microporosity, osteoclastic degradation of the material was inhibited. Thus, even for wide-open porous TCP-based scaffolds, a high degree of microporosity appears to be essential for optimal osteoconduction and creeping substitution, which can prevent non-unions, the major complication during bone regeneration procedures.

Aim: To test whether or not bone regeneration using deproteinized bovine bone mineral (DBBM) is comparable to hydroxyapatite/silica oxide (HA/SiO) and to test the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) as an adjunct to DBBM for localized bone regeneration., Materials and Methods: In each of the 10 rabbits, 4 titanium cylinders were placed on the external cortical plates of their calvaria. Four treatment modalities were randomly allocated: (i) empty, (ii) HA/SiO, (iii) DBBM, and (iv) DBBM plus rhBMP-2 (DBBM/BMP). The animals were sacrificed at week 8. Descriptive histology and histomorphometric assessment using a superimposed test grid of points and cycloids were performed., Results: The mean number of points of the test grid coinciding with bone within the cylinder reached 124 ± 35 bone points for empty controls, 92 ± 40 bone points for DBBM, 98 ± 44 bone points for synthetic HA/SiO, and 146 ± 34 bone points DBBM/BMP. The P-value for DBBM with and without BMP reached a borderline statistical significance of 0.051. However, the area of bone regeneration within the cylinders peaked for DBBM/BMP and was statistically significantly higher compared with empty cylinders (P < 0.05). The bone-to-bone substitute contact ranged between 32.9% ± 21.7 for DBBM, 39.6 ± 18.4% for HA/SiO, and 57.8% ± 10.2 for DBBM/BMP. The differences between DBBM/BMP and controls (DBBM, HA/SiO) were statistically significant (P < 0.05)., Conclusions: DBBM and HA/SiO rendered comparable amounts of bone regeneration. The addition of rhBMP-2 to DBBM resulted in more favorable outcomes with respect to the area of bone regeneration and to bone-to-implant contact, thereby indicating the potential of this growth factor to enhance bone regeneration within this animal model., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Bone morphogenetic proteins (BMPs) are deposited in bone and responsible for osteoinduction. The interplay between delivery system and BMP, resulting in a characteristic release profile, is crucial for clinical success. We here report on two apatite based commercially available granules which could potentially be used in a combination product with recombinant human BMP-2 (rhBMP-2). Regardless of their similar chemistry, their interaction with rhBMP-2 differs. Deproteinized bovine bone matrix (DBBM), a clinically well-established bone substitute, has a high affinity to rhBMP-2 and releases only 50% of the growth factor during the first 2 weeks in vitro. Activity of the physio-adsorbed rhBMP-2 is indicated by an enhanced bone augmentation in vivo. In contrast, all rhBMP-2 delivered in combination with synthetic hydroxyapatite/β-tricalcium phosphate (HA/TCP) granules is released during the first 24 h. For both HA/TCP and DBBM, the released rhBMP-2 is active in vitro. Our results suggest that the different release behavior from these two apatite granules is due to the 1000-fold higher specific surface area of DBBM compared to HA/TCP., (© 2014 Wiley Periodicals, Inc.)

The treatment of large bone defects still poses a major challenge in orthopaedic and cranio-maxillofacial surgery. One possible solution could be the development of personalized porous titanium-based implants that are designed to meet all mechanical needs with a minimum amount of titanium and maximum osteopromotive properties so that it could be combined with growth factor-loaded hydrogels or cell constructs to realize advanced bone tissue engineering strategies. Such implants could prove useful for mandibular reconstruction, spinal fusion, the treatment of extended long bone defects, or to fill in gaps created on autograft harvesting. The aim of this study was to determine the mechanical properties and potential of bone formation of light weight implants generated by selective laser melting (SLM). We mainly focused on osteoconduction, as this is a key feature in bone healing and could serve as a back-up for osteoinduction and cell transplantation strategies. To that end, defined implants were produced by SLM, and their surfaces were left untreated, sandblasted, or sandblasted/acid etched. In vivo bone formation with the different implants was tested throughout calvarial defects in rabbits and compared with untreated defects. Analysis by micro computed tomography (μCT) and histomorphometry revealed that all generatively produced porous Ti structures were well osseointegrated into the surrounding bone. The histomorphometric analysis revealed that bone formation was significantly increased in all implant-treated groups compared with untreated defects and significantly increased in sand blasted implants compared with untreated ones. Bone bridging was significantly increased in sand blasted acid-etched scaffolds. Therefore, scaffolds manufactured by SLM should be surface treated. Bone augmentation beyond the original bone margins was only seen in implant-treated defects, indicating an osteoconductive potential of the implants that could be utilized clinically for bone augmentation purposes. Therefore, designed porous, lightweight structures have potential for bone regeneration and augmentation purposes, especially when complex and patient-specific geometries are essential.

Objective: Moldable in situ self-stabilizing and hardening bone graft materials facilitate handling and may be suitable for membrane-free bone regeneration methods. This study aimed to compare two moldable synthetic calcium phosphate materials in a rabbit calvarial defect model., Method: In 12 New Zealand white rabbits, four evenly distributed 6 mm diameter defects were drilled in the calvarial bone. Three filler materials were randomly applied to 48 defects: an in situ hardening polylactide-coated ß-tricalcium phosphate (TCP), an in situ hardening polylactide-coated biphasic calcium phosphate (BCP), and a granular deproteinized bovine bone matrix (DBBM, positive control). One defect remained untreated and served as a negative control. Six animals were sacrificed after 4 weeks, and the remaining animals were sacrificed after 16 weeks. Biocompatibility, bone graft substitute integration and resorption, bone formation, defect bridging, and height of reconstructed hard tissue were assessed histologically and histomorphometrically., Results: All tested materials showed good biocompatibility. Semi-quantitative analysis and pair-wise comparison suggested that BCP was more efficient in centripetal bone formation when compared with TCP. After 4 weeks, significantly more bone had formed in the defects treated with either TCP or BCP materials compared with the untreated sites. BCP and DBBM did not show macroscopic signs of degradation, whereas the TCP material was partially resorbed after 16 weeks. Otherwise, no major differences were detected between the three materials., Conclusion: The moldable, synthetic calcium phosphates are safe and suitable bone graft substitutes with outcomes that are comparable to the control material., (© 2011 John Wiley & Sons A/S.)

Objectives: A comparison of synthetic hydroxyapatite/silica oxide, xenogenic hydroxyapatite-based bone substitute materials with empty control sites in terms of bone regeneration enhancement in a rabbit calvarial four non-critical-sized defect model., Methods: In each of six rabbits, four bicortical calvarial bone defects were generated. The following four treatment modalities were randomly allocated: (1) empty control site, (2) synthetic hydroxyapatite/silica oxide-based (HA/SiO) test granules, (3) xenogenic hydroxyapatite -based granules, (4) synthetic hydroxyapatite/silica oxide -based (HA/SiO) test two granules. The results of the latter granules have not been reported due to their size being three times bigger than the other two granule types. After 4 weeks, the animals were sacrificed and un-decalcified sections were obtained for histological analyses. For statistical analysis, the Kruskal-Wallis test was applied (P<0.05)., Results: Histomorphometric analysis showed an average area fraction of newly formed bone of 12.32±10.36% for the empty control, 17.47±6.42% for the xenogenic hydroxyapatite -based granules group, and 21.2±5.32% for the group treated with synthetic hydroxyapatite/silica oxide -based granules. Based on the middle section, newly formed bone bridged the defect to 38.33±37.55% in the empty control group, 54.33±22.12% in the xenogenic hydroxyapatite -based granules group, and to 79±13.31% in the synthetic hydroxyapatite/silica oxide -based granules group. The bone-to-bone substitute contact was 46.38±18.98% for the xenogenic and 59.86±14.92% for the synthetic hydroxyapatite/silica oxide-based granules group. No significant difference in terms of bone formation and defect bridging could be detected between the two bone substitute materials or the empty defect., Conclusion: There is evidence that the synthetic hydroxyapatite/silica oxide granules provide comparable results with a standard xenogenic bovine mineral in terms of bone formation and defect bridging in non-critical size defects., (© 2010 John Wiley & Sons A/S.)

Purpose: The aim of the present study was to test whether or not a synthetic matrix consisting of a polyethylene glycol (PEG) hydrogel containing recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with grafting materials enhances bone regeneration compared with grafting alone or empty control sites., Material and Methods: In each of 10 rabbits, four titanium cylinders were screwed in perforated slits made in the external cortical bones of the calvaria. The following four treatment modalities were randomly allocated: (1) empty control, (2) a combination of a PEG matrix and hydroxyapatite/tricalciumphosphate (HA/TCP) granules and a combination of a PEG matrix containing either 10 microg/ml (3) or 30 microg/ml (4) of BMP-2 and HA/TCP granules. After 8 weeks, the animals were sacrificed and ground sections were obtained for histological analysis. For statistical analysis repeated measures ANOVA and subsequent pairwise Student's t-test were applied (P<0.01)., Results: Histomorphometric analysis showed an average area fraction of newly formed bone of 13.96+/-5.98% for the empty control, 15.16+/-7.95% for the PEG and HA/TCP group, 26.32+/-8.56% for the group containing 10 mug rhBMP-2/ml, and 30.15+/-7.63% for the group containing 30 microg rhBMP-2/ml. Statistical analysis revealed significantly more newly formed bone in the two rhBMP-2 groups compared with the PEG and HA/TCP group and with the empty control. Regarding the surface fraction of the HA/TCP graft particles covered with newly formed bone the addition of rhBMP-2 revealed a more than two-fold increase compared with cylinders containing HA/TCP granules without rhBMP-2. This difference reached statistical significance., Conclusions: It is concluded that rhBMP-2 significantly enhances bone regeneration in rabbits when delivered by a synthetic matrix containing HA/TCP. This synthetic PEG matrix containing HA/TCP granules apparently fulfills a number of criteria required for an ideal carrier system for rhBMP-2.

Purpose: The aim of the present study was to test whether a newly developed synthetic matrix made of polyethylene-glycol (PEG) containing a covalently bound peptide of the parathyroid hormone (PTH1-34) enhances bone regeneration compared to grafting procedures and to spontaneous healing., Materials and Methods: In each of 16 rabbits used, 4 titanium cylinders were screwed into perforated slits made in the cortical bone of the calvaria. The cylinders were either left empty (control) or filled with 1 of the following: (1) PEG matrix and hydroxyapatite/tricalcium phosphate (HA/TCP) granules, (2) PEG matrix containing 100microg/mL of PTH and HA/TCP granules, or (3) PEG matrix containing 20microg/mL of PTH1-34 and HA/TCP granules. After 8 weeks, the animals were sacrificed, and ground sections were obtained for histology., Results: Quantitative histomorphometry demonstrated a significantly increased amount of newly formed bone for PTH1-34 compared to sites treated with PEG and HA/TCP and to empty control sites (P < .01; analysis of variance and subsequent pairwise Student t test). The mean percentages of mineralized bone were 19.6%+/-6.0% for 100 microg/mL PTH, 18.0% +/- 6.2% for 20microg/mL PTH, 12.0% +/-6.5% for PEG and HA/TCP without PTH, and 10.5% +/-3.7% for the empty control. The mean areas of bone regenerated within the cylinders were 53.5% +/-22.7% for 100 microg/mL PTH, 51.1% +/-22.6% for 20 microg/mL PTH, 34.3% +/- 22.5% for PEG and HA/TCP without PTH, and 23.2% +/-10.1% for the empty control., Discussion: Human and animal trials have demonstrated that daily systemic injection of PTH increases bone mineral density. The present study showed that local administration of PTH was also effective in stimulating bone formation., Conclusion: It is concluded that this synthetic PEG hydrogel containing a covalently bound peptide of the PTH combined with HA/TCP granules significantly stimulated in situ bone augmentation in rabbits.

Cell interactions with the extracellular matrix play important roles in guiding tissue morphogenesis. The matrix stimulates cells to influence such things as differentiation and the cells actively remodel the matrix via local proteolytic activity. We have designed synthetic hydrogel networks that participate in this interplay: They signal cells via bound adhesion and growth factors, and they also respond to the remodeling influence of cell-associated proteases. Poly(ethylene glycol)-bis-vinylsulfone was crosslinked by a Michael-type addition reaction with a peptide containing three cysteine residues, the peptide sequence being cleavable between each cysteine residue by the cell-associated protease plasmin. Cells were able to invade gel networks that contained adhesion peptides and were crosslinked by plasmin-sensitive peptides, while materials lacking either of these two characteristics resisted cell infiltration. Incorporated bone morphogenetic protein-2 (BMP-2) induced bone healing in a rat model in materials that were both adhesive and plasmin-sensitive, while materials lacking plasmin sensitivity resisted formation of bone within the material. Furthermore, when a heparin bridge was incorporated as a BMP-2 affinity site, mimicking yet another characteristic of the extracellular matrix, statistically improved bone regeneration was observed., (Copyright 2004 Wiley Periodicals, Inc.)

We have engineered synthetic poly(ethylene glycol) (PEG)-based hydrogels as cell-ingrowth matrices for in situ bone regeneration. These networks contain a combination of pendant oligopeptide ligands for cell adhesion (RGDSP) and substrates for matrix metalloproteinase (MMP) as linkers between PEG chains. Primary human fibroblasts were shown to migrate within these matrices by integrin- and MMP-dependent mechanisms. Gels used to deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) to the site of critical- sized defects in rat crania were completely infiltrated by cells and were remodeled into bony tissue within five weeks. Bone regeneration was dependent on the proteolytic sensitivity of the matrices and their architecture. The cell-mediated proteolytic invasiveness of the gels and entrapment of rhBMP-2 resulted in efficient and highly localized bone regeneration.

The application of autogenous bone transplants is still the gold standard for all reconstructive surgery. The wish of surgeons for alternatives to autogenous bone grafts is reflected in the development of a variety of bone graft substitutes of synthetic or biological origin. The following review article is meant to provide the reader with a critical overview of all the alternative materials. In addition, we want to point to the tremendous progress made on this subject, made possible by the joint effort of surgeons and basic researchers. The results of this collaboration show that in the near future a new gold standard for successful reconstructive surgery will emerge and replace the use of autogenous bone transplants.