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Osteoinductive materials are characterized by their ability to induce bone formation in ectopic sites. Thus, osteoinductive materials hold promising potential for repairing bone defects. However, the mechanism of material-induced bone formation remains unknown, which limits the design of highly potent osteoinductive materials. Here, we demonstrated a genetic background link among macrophage polarization, osteoclastogenesis and material-induced bone formation. The intramuscular implantation of an osteoinductive material in FVB/NCrl (FVB) mice resulted in more M2 macrophages at week 1, more osteoclasts at week 2 and increased bone formation after week 4 compared with the results obtained in C57BL/6JOlaHsd (C57) mice. Similarly, in vitro, with a greater potential to form M2 macrophages, monocytes derived from FVB mice formed more osteoclasts than those derived from C57 mice. A transcriptomic analysis identified Csf1, Cxcr4 and Tgfbr2 as the main genes controlling macrophage-osteoclast coupling, which were further confirmed by related inhibitors. With such coupling, macrophage polarization and osteoclast formation of monocytes in vitro successfully predicted in vivo bone formation in four other mouse strains. Considering material-induced bone formation as an example of acquired heterotopic bone formation, the current findings shed a light on precision medicine for both bone regeneration and the treatment of pathological heterotopic bone formation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Heterotopic ossification (HO) is a double-edged sword. Pathological HO presents as an undesired clinical complication, whereas controlled heterotopic bone formation by synthetic osteoinductive materials shows promising therapeutic potentials for bone regeneration. However, the mechanism of material-induced heterotopic bone formation remains largely unknown. Early acquired HO being usually accompanied by severe tissue hypoxia prompts the hypothesis that hypoxia caused by the implantation coordinates serial cellular events and ultimately induces heterotopic bone formation in osteoinductive materials. The data presented herein shows a link between hypoxia, macrophage polarization to M2, osteoclastogenesis, and material-induced bone formation. Hypoxia inducible factor-1α (HIF-1α), a crucial mediator of cellular responses to hypoxia, is highly expressed in an osteoinductive calcium phosphate ceramic (CaP) during the early phase of implantation, while pharmacological inhibition of HIF-1α significantly inhibits M2 macrophage, subsequent osteoclast, and material-induced bone formation. Similarly, in vitro, hypoxia enhances M2 macrophage and osteoclast formation. Osteoclast-conditioned medium enhances osteogenic differentiation of mesenchymal stem cells, such enhancement disappears with the presence of HIF-1α inhibitor. Furthermore, metabolomics analysis reveals that hypoxia enhances osteoclastogenesis via the axis of M2/lipid-loaded macrophages. The current findings shed new light on the mechanism of HO and favor the design of more potent osteoinductive materials for bone regeneration., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Objectives: The aim of this study was the preclinical and clinical evaluation of osteoinductive calcium phosphate with submicron surface topography as a bone graft substitute for maxillary sinus floor augmentation (MSFA)., Material and Methods: A preclinical sheep model of MSFA was used to compare a calcium phosphate with submicron needle-shaped topography (BCP N , MagnetOs Granules, Kuros Biosciences BV) to a calcium phosphate with submicron grain-shaped topography (BCP G ) and autologous bone graft (ABG) as controls. Secondly, a 10-patient, prospective, randomized, controlled trial was performed to compare BCP N to ABG in MSFA with two-stage implant placement., Results: The pre-clinical study demonstrated that both BCP N and BCP G were highly biocompatible, supported bony ingrowth with direct bone apposition against the material, and exhibited bone formation as early as 3 weeks post-implantation. However, BCP N demonstrated significantly more bone formation than BCP G at the study endpoint of 12 weeks. Only BCP N reached an equivalent amount of bone formation in the available space and a greater proportion of calcified material (bone + graft material) in the maxillary sinus compared to the "gold standard" ABG after 12 weeks. These results were validated in a small prospective clinical study, in which BCP N was found comparable to ABG in implant stability, bone height, new bone formation in trephine core biopsies, and overall clinical outcome., Conclusion: This translational work demonstrates that osteoinductive calcium phosphates are promising bone graft substitutes for MSFA, whereas their bone-forming potential depends on the design of their surface features. Netherlands Trial Register, NL6436., (© 2023 The Authors. Clinical Oral Implants Research published by John Wiley & Sons Ltd.)

Innate immune responses play important roles in material-induced bone formation and such roles were further explored in the current study with an emphasis on M2 macrophages and osteoclastogenesis. With the presence of M-CSF and RANKL, M0 macrophages from FVB mouse bone marrow-derived monocytes (BMMs) fused to osteoclasts with both M2 marker and osteoclast marker at day 5, and such osteoclast formation at day 5 was enhanced when the cells were treated with IL-4 at day 3. With IL-4 treatment alone for 24 h, M0 polarized into M2 macrophages. Conditioned medium of M2 macrophages enhanced osteogenic differentiation of MC3T3-E1 (pre-osteoblasts) while osteoclast conditioned medium enhanced osteogenic differentiation of CRL-12424 (osteogenic precursors). TCPs (a typical osteoinductive material) supported M2 macrophage polarization at day 4 and osteoclast formation at day 5, while TCPb (a typical non-osteoinductive material) was less effective. Moreover, osteoclasts formed on TCPs produced osteogenic factors including S1P, Wnt10B and BMP-6, resulting osteogenic differentiation of CRL-12424 cells. Similar to in vitro testing, TCPs favored M2 macrophage polarization followed by the formation of osteoclasts in vivo, as compared to TCPb. The overall data provided evidence of a coupling between M2 macrophages, osteoclasts and material-induced bone formation: osteoclasts formed from M2 macrophages secrete osteogenic cytokines to induce osteogenic differentiation of osteogenic precursor cells to finally form bone. The current findings outlined a biological mechanism of material-induced bone formation and further rationalized the use of osteoinductive materials for bone regeneration. STATEMENT OF SIGNIFICANCE: This paper provides evidence for finding out the relationship between M2 macrophages, osteoclasts and osteogenesis in material-induced bone formation. It suggested that osteoinductive materials enhanced macrophage polarization to M2 macrophages which fuses to osteoclasts, osteoclasts subsequently secret osteogenic cytokines to differentiate finally osteogenic precursors to form bone in osteoinductive materials. The data supports scientifically the superiority of osteoinductive materials for bone regeneration in clinics., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2022. Published by Elsevier Ltd.)

A biphasic calcium phosphate with submicron needle-shaped surface topography combined with a novel polyethylene glycol/polylactic acid triblock copolymer binder (BCP-EP) was investigated in this study. This study aims to evaluate the composition, degradation mechanism and bioactivity of BCP-EP in vitro, and its in vivo performance as an autograft bone graft (ABG) extender in a rabbit Posterolateral Fusion (PLF) model. The characterization of BCP-EP and its in vitro degradation products showed that the binder hydrolyses rapidly into lactic acid, lactide oligomers and unaltered PEG (polyethylene glycol) without altering the BCP granules and their characteristic submicron needle-shaped surface topography. The bioactivity of BCP-EP after immersion in SBF revealed a progressive surface mineralization. In vivo, BCP-EP was assessed in a rabbit PLF model by radiography, manual palpation, histology and histomorphometry up to 12 weeks post-implantation. Twenty skeletally mature New Zealand (NZ) White Rabbits underwent single-level intertransverse process PLF surgery at L4/5 using (1) autologous bone graft (ABG) alone or (2) by mixing in a 1:1 ratio with BCP-EP (BCP-EP/ABG). After 3 days of implantation, histology showed the BCP granules were in direct contact with tissues and cells. After 12 weeks, material resorption and mature bone formation were observed, which resulted in solid fusion between the two transverse processes, following all assessment methods. BCP-EP/ABG showed comparable fusion rates with ABG at 12 weeks, and no graft migration or adverse reaction were noted at the implantation site nor in distant organs.

To better understand the biological mechanisms triggered by osteoinductive materials in vivo, we evaluated the timeline of cellular responses to osteoinductive materials subcutaneously implanted in FVB mice. More F4/80-positive macrophages were present in osteoinductive tri-CaP ceramic (TCP) with submicron surface topography (TCPs) than non-osteoinductive TCP with micron surface topography (TCPb) at week 1. Moreover, TCPs (but not TCPb) significantly enhanced osteoclastogenesis, and induced macrophages to polarize from M1 to M2 in the first week. The time sequence and relevance of macrophages and osteoclasts responses involved in bone formation was then evaluated through peri-implant injection of specific chemicals in mice implanted with osteoinductive TCPs. Day-1 injection of clodronate liposomes (LipClod) depleted macrophages, inhibited macrophage polarization to M2, blocked osteoclastogenesis and bone formation, while the day-6 injection was less effective. Anti-RANKL antibody (aRANKL) did not affect macrophage colonization but inhibited osteoclastogenesis. Injection of aRANKL before week 2 aborted bone formation in TCPs, while injection at week 4 partially inhibited bone formation. The overall data show that following ectopic implantation, osteoinductive materials allow macrophage colonization in hours to days, macrophage polarization to M2 in days (within 7 days), osteoclastogenesis in weeks (e.g. in 2 weeks) and bone formation thereafter (after 4 weeks). The serial cellular events verified herein bring a new insight on material-induced bone formation and pave the way to further explore the mechanisms triggered by osteoinductive materials. STATEMENT OF SIGNIFICANCE: A series of key cellular events triggered by osteoinductive calcium phosphate ceramic was revealed: macrophages colonized within hours to days, polarization of M2 macrophages occurred within 7 days, osteoclastogenesis mainly occurred in weeks (e.g. in 2 weeks) and bone formation finally arose thereafter (after 4 weeks). Moreover, such time sequence of cellular events was confirmed with specific chemicals (clodronate liposomes and anti-RANKL antibody). The findings verified herein bring a new insight on material-induced bone formation and pave the way to further explore the mechanisms triggered by osteoinductive materials., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2021. Published by Elsevier Ltd.)

Proper regulation of the innate immune response to bone biomaterials after implantation is pivotal for successful bone healing. Pro-inflammatory M1 and anti-inflammatory M2 macrophages are known to have an important role in regulating the healing response to biomaterials. Materials with defined structural and topographical features have recently been found to favourably modulate the innate immune response, leading to improved healing outcomes. Calcium phosphate bone grafts with submicron-sized needle-shaped surface features have been shown to trigger a pro-healing response through upregulation of M2 polarised macrophages, leading to accelerated and enhanced bone regeneration. The present review describes the recent research on these and other materials, all the way from benchtop to the clinic, including in vitro and in vivo fundamental studies, evaluation in clinically relevant spinal fusion models and clinical validation in a case series of 77 patients with posterolateral and/or interbody fusion in the lumbar and cervical spine. This research demonstrates the feasibility of enhancing biomaterial-directed bone formation by modulating the innate immune response through topographic surface features.

Chimeric antigen receptor (CAR) T cells are highly successful in the treatment of hematologic malignancies. We recently generated affinity-optimized CD38CAR T cells, which effectively eliminate multiple myeloma (MM) cells with little or no toxicities against nonmalignant hematopoietic cells. The lack of universal donors and long manufacturing times however limit the broad application of CAR T cell therapies. Natural killer (NK) cells generated from third party individuals may represent a viable source of "off the shelf" CAR-based products, as they are not associated with graft-versus-host disease unlike allogeneic T cells. We therefore explored the preclinical anti-MM efficacy and potential toxicity of the CD38CAR NK concept by expressing affinity-optimized CD38CARs in KHYG-1 cells, an immortal NK cell line with excellent expansion properties. KHYG-1 cells retrovirally transduced with the affinity-optimized CD38CARs expanded vigorously and mediated effective CD38-dependent cytotoxicity towards CD38 high MM cell lines as well as primary MM cells ex vivo. Importantly, the intermediate affinity CD38CAR transduced KHYG-1 cells spared CD38 neg or CD38 int nonmalignant hematopoietic cells, indicating an optimal tumor nontumor discrimination. Irradiated, short living CD38CAR KHYG-1 cells also showed significant anti-MM effects in a xenograft model with a humanized bone marrow-like niche. Finally, CD38CAR KHYG-1 cells effectively eliminated primary MM cells derived from patients who are refractory to CD38 antibody daratumumab. Taken together, the results of this proof-of-principle study demonstrate the potential value of engineering affinity-optimized CD38CARs in NK cells to establish effective anti-MM effects, with an excellent safety profile, even in patients who failed to response to most advanced registered myeloma therapies, such as daratumumab., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Study Design: This study was a multi-endpoint analysis of bone graft substitutes implanted as a standalone graft in a clinically relevant Ovine model of instrumented posterolateral spinal fusion (PLF)., Objective: The objective of this study was to obtain high-quality evidence on the efficacy of commercial bone graft substitutes compared with autograft in instrumented PLF using a state-of-the-art model with a complete range of assessment techniques., Summary of Background Data: Preclinical and clinical data on the quality of spinal fusions obtained with bone graft substitutes are often limited. Calcium phosphates with submicron topography have shown promising results in PLF, as these are able to induce bone formation in tissues distant from the host bone, which facilitates bony union., Methods: Nine female, skeletally mature sheep (4-5 y) underwent posterior pedicle screw/rods instrumented PLF at L2-L3 and L4-L5 using the following bone graft materials as a standalone graft per spinal segment: (1) biphasic calcium phosphate with submicron topography (BCP<µm), (2) 45S5 Bioglass (BG), and (3) collagen-β-tricalcium phosphate with a 45S5 Bioglass adjunct (TCP/BG). Autograft bone (AB) was used as a positive control treatment. Twelve weeks after implantation, the spinal segments were evaluated by fusion assessment (manual palpation, x-ray, micro-computed tomography, and histology), fusion mass volume quantification (micro-computed tomography), range of motion (ROM) testing, histologic evaluation, and histomorphometry., Results: Fusion assessment revealed equivalence between AB and BCP<µm by all fusion assessment methods, whereas BG and TCP/BG led to significantly inferior results. Fusion mass volume was highest for BCP<µm, followed by AB, BG, and TCP/BG. ROM testing determined equivalence for spinal levels treated with AB and BCP<µm, while BG and TCP/BG exhibited higher ROM. Histologic evaluation revealed substantial bone formation in the intertransverse regions for AB and BCP<µm, whereas BG and TCP/BG grafts contained fibrous tissue and minimal bone formation. Histologic observations were supported by the histomorphometry data., Conclusions: This study reveals clear differences in efficacy between commercially available bone graft substitutes, emphasizing the importance of clinically relevant animal models with multiendpoint analyses for the evaluation of bone graft materials. The results corroborate the efficacy of calcium phosphate with submicron topography, as this was the only material that showed equivalent performance to autograft in achieving spinal fusion.

To investigate the roles of macrophages in material-instructed bone formation, two calcium phosphate (TCP) ceramics with the same chemistry but various scales of surface topography were employed in this study. After being implanted subcutaneously in FVB mice for 8 weeks, TCPs (TCP ceramics with submicron surface topography) gave rise to bone formation, while TCPb (TCP ceramics with micron surface topography) did not, showing the crucial role of surface topography scale in material-instructed bone formation. Depletion of macrophages with liposomal clodronate (LipClod) blocked such bone formation instructed by TCPs, confirming the role of macrophages in material-instructed bone formation. Macrophage cells (i.e. RAW 264.7 cells) cultured on TCPs in vitro polarized to tissue repair macrophages as evidenced by gene expression and cytokine production, while polarizing to pro-inflammatory macrophages on TCPb. Submicron surface topography of TCP ceramics directed macrophage polarization via PI3K/AKT pathways with the synergistic regulation of integrin β1. Finally, the tissue repair macrophage polarization on TCPs resulted in osteogenic differentiation of mesenchymal stem cells in vitro. At early implantation in FVB mice, TCPs recruited more macrophages which polarized towards tissue repair macrophages with time. The present data demonstrate the important roles of macrophage polarization in bone formation instructed by calcium phosphate ceramics.

Multiple myeloma is characterized by accumulation of malignant plasma cells in the bone marrow. Most patients suffer from an osteolytic bone disease, caused by increased bone degradation and reduced bone formation. Bone morphogenetic protein 4 (BMP4) is important for both pre- and postnatal bone formation and induces growth arrest and apoptosis of myeloma cells. BMP4-treatment of myeloma patients could have the potential to reduce tumor growth and restore bone formation. We therefore explored BMP4 gene therapy in a human-mouse model of multiple myeloma where humanized bone scaffolds were implanted subcutaneously in RAG2 -/- γC -/- mice. Mice were treated with adeno-associated virus serotype 8 BMP4 vectors (AAV8-BMP4) to express BMP4 in the liver. When mature BMP4 was detectable in the circulation, myeloma cells were injected into the scaffolds and tumor growth was examined by weekly imaging. Strikingly, the tumor burden was reduced in AAV8-BMP4 mice compared with the AAV8-CTRL mice, suggesting that increased circulating BMP4 reduced tumor growth. BMP4-treatment also prevented bone loss in the scaffolds, most likely due to reduced tumor load. To delineate the effects of BMP4 overexpression on bone per se, without direct influence from cancer cells, we examined the unaffected, non-myeloma femurs by μCT. Surprisingly, the AAV8-BMP4 mice had significantly reduced trabecular bone volume, trabecular numbers, as well as significantly increased trabecular separation compared with the AAV8-CTRL mice. There was no difference in cortical bone parameters between the two groups. Taken together, BMP4 gene therapy inhibited myeloma tumor growth, but also reduced the amount of trabecular bone in mice. Our data suggest that care should be taken when considering using BMP4 as a therapeutic agent. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research., (© 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.)

Posterolateral spinal fusion (PLF) is a common procedure in orthopedic surgery that is performed to fuse adjacent vertebrae to reduce symptoms related to spinal conditions. In the current study, a novel synthetic calcium phosphate with submicron surface topography was evaluated as an autograft extender in a validated rabbit model of PLF. Fifty-nine skeletally mature New Zealand white rabbits were divided into three groups and underwent single-level intertransverse process PLF at L4-5 using (1) autologous bone graft (ABG) alone or in a 1:1 combination with (2) calcium phosphate granules (ABG/BCP granules ), or (3) granules embedded in a fast-resorbing polymeric carrier (ABG/BCP putty ). After 6, 9, and 12 weeks, animals were sacrificed and spinal fusion was assessed by manual palpation, Radiographs, micro-CT, mechanical testing (12 weeks only), histology, and histomorphometry. Based on all endpoints, all groups showed a gradual progression in bone formation and maturation during time, leading to solid fusion masses between the transverse processes after 12 weeks. Fusion assessments by manual palpation, radiography and histology were consistent and demonstrated equivalent fusion rates between groups, with high bilateral fusion rates after 12 weeks. Mechanical tests after 12 weeks indicated substantially lower range of motion for all groups, compared to non-operated controls. By histology and histomorphometry, the gradual formation and maturation of bone in the fusion mass was confirmed for each graft type. With these results, we describe the equivalent performance between autograft and a novel calcium phosphate material as an autograft extender in a rabbit model of PLF using an extensive range of evaluation techniques. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2080-2090, 2019., (© 2019 Wiley Periodicals, Inc.)

Background: Cell-based therapies have the potential to become treatment options for many diseases, but efficient scale-out of these therapies has proven to be a major hurdle. Bioreactors can be used to overcome this hurdle, but changing the culture method can introduce unwanted changes to the cell product. Therefore, it is important to establish parity between products generated using traditional methods versus those generated using a bioreactor., Methods: Mesenchymal stromal cells (MSCs) are cultured in parallel using either traditional culture flasks, spinner vessels or a new bioreactor system. To investigate parity between the cells obtained from different methods, harvested cells are compared in terms of yield, phenotype and functionality., Results: Bioreactor-based expansion yielded high cell numbers (222-510 million cells). Highest cell expansion was observed upon culture in flasks [average 5.0 population doublings (PDL)], followed by bioreactor (4.0 PDL) and spinner flasks (3.3 PDL). Flow cytometry confirmed MSC identity (CD73 + , CD90 + and CD105 + ) and lack of contaminating hematopoietic cell populations. Cultured MSCs did not display genetic aberrations and no difference in differentiation and immunomodulatory capacity was observed between culture conditions. The response to IFNγ stimulation was similar for cells obtained from all culture conditions, as was the capacity to inhibit T cell proliferation., Conclusions: The new bioreactor technology can be used to culture large amounts of cells with characteristics equivalent to those cultured using traditional, flask based, methods.

Osteoinductive calcium phosphate (CaP) bone grafts have equivalent performance to autografts in repairing critical-size bone defects. The osteoinductive potential of CaP is linked to the size of the surface topographical features. In the present study, two novel biphasic calcium phosphate (BCP) bone grafts were synthesised with either sub-micron- (BCP<µm) or micron-scale (BCPµm) needle-shaped surface topography and compared to dimensionally similar tricalcium phosphate (TCP) with grain-shaped surface structures (TCP<µm and TCPµm). To clarify the possible function of the surface morphology (needle-like vs. grain-like) in initiating bone formation, the four CaP test materials were physicochemically characterised and implanted for 12 weeks in the dorsal muscle of beagles. The sub-micron needle-shaped topography of BCP<µm triggered earlier bone formation (3-6 weeks) as compared to the grain-shaped surface topography of TCP<µm, which formed bone at 6-9 weeks. After 12 weeks, the amount of induced bone formation in both materials was equivalent, based on histomorphometry. The micron-sized needle-shaped surface topography of BCPµm led to limited formation of new bone tissue, whereas its counterpart, TCPµm with grain-shaped surface topography, failed to trigger de novo bone formation. The relative strength of the parameters affecting CaP-driven bone induction was as follows: surface feature size > surface feature morphology > substrate chemistry. BCP materials with needle-shaped sub-micron surface topography gave rise to accelerated bone formation and slower rate of resorption than a comparable TCP. These characteristics may be translated to improve bone healing in orthotopic defects.

As spinal fusions require large volumes of bone graft, different bone graft substitutes are being investigated as alternatives. A subclass of calcium phosphate materials with submicron surface topography has been shown to be a highly effective bone graft substitute. In this work, a commercially available biphasic calcium phosphate (BCP) with submicron surface topography (MagnetOs; Kuros Biosciences BV) was evaluated in an Ovine model of instrumented posterolateral fusion. The material was implanted stand-alone, either as granules (BCP granules ) or as granules embedded within a fast-resorbing polymeric carrier (BCP putty ) and compared to autograft bone (AG). Twenty-five adult, female Merino sheep underwent posterolateral fusion at L2-3 and L4-5 levels with instrumentation. After 6, 12, and 26 weeks, outcomes were evaluated by manual palpation, range of motion (ROM) testing, micro-computed tomography, histology and histomorphometry. Fusion assessment by manual palpation 12 weeks after implantation revealed 100% fusion rates in all treatment groups. The three treatment groups showed a significant decrease in lateral bending at the fusion levels at 12 weeks ( P  < 0.05) and 26 weeks ( P  < 0.001) compared to the 6 week time-point. Flexion-extension and axial rotation were also reduced over time, but statistical significance was only reached in flexion-extension for AG and BCP putty between the 6 and 26 week time-points ( P  < 0.05). No significant differences in ROM were observed between the treatment groups at any of the time-points investigated. Histological assessment at 12 weeks showed fusion rates of 75%, 92%, and 83% for AG, BCP granules and BCP putty , respectively. The fusion rates were further increased 26 weeks postimplantation. Similar trends of bone growth were observed by histomorphometry. The fusion mass consisted of at least 55% bone for all treatment groups 26 weeks after implantation. These results suggest that this BCP with submicron surface topography, in granules or putty form, is a promising alternative to autograft for spinal fusion., Competing Interests: This study was supported by Kuros Biosciences BV. L. van D., R.D., X.L., D.B., F.B. de G., H.Y., and J.D. de B. are employees, or former employees, of Kuros Biosciences BV. H.Y. and J.D. de B. are stockholders of Kuros Biosciences BV. C.C., M.P., A.R., and W.W. have nothing to disclose.

Tricalcium phosphate (TCP) ceramics are used as bone void fillers because of their bioactivity and resorbability, while their performance in bone regeneration and material resorption vary with their physical properties (e.g., the dimension of the crystal grain). Herein, three TCP ceramic bone substitutes (TCP-S, TCP-M, and TCP-L) with gradient crystal grain size (0.77 ± 0.21 μm for TCP-S, 1.21 ± 0.35 μm for TCP-M and 4.87 ± 1.90 μm for TCP-L), were evaluated in a well-established rabbit lateral condylar defect model (validated with sham) with respect to bone formation and material resorption up to 26 weeks. Surface structure-dependent bone regeneration was clearly shown after 4 weeks implantation with TCP-S having most mineralized bone (20.2 ± 3.4%), followed by TCP-M (14.0 ± 3.5%), sham (8.1 ± 4.2%), and TCP-L (6.6 ± 2.6%). Afterward, the amount of mineralized bone was similar in all the three groups, but bone marrow and material resorption varied. After 26 weeks, TCP-S induced most bone tissue formation (mineralized bone + bone marrow) (61.6 ± 7.8%) and underwent most material resorption (80.1 ± 9.0%), followed by TCP-M (42.9 ± 5.2% and 61.4 ± 8.0% respectively), TCP-L (28.3 ± 5.5% and 45.6 ± 9.7% respectively), and sham (25.7 ± 4.2%). Given the fact that the three ceramics are chemically identical, the results indicate that the surface structure (especially, the crystal grain size) of TCP ceramics can greatly tune their bone regeneration potential and the material resorption in rabbit condyle defect model, with the submicron surface structured TCP ceramic performing the best., Competing Interests: The authors declare no competing financial interest.

Because of their bioactive properties and chemical similarity to the inorganic component of bone, calcium phosphate (CaP) materials are widely used for bone regeneration. Six commercially available CaP bone substitutes (Bio-Oss, Actifuse, Bi-Ostetic, MBCP, Vitoss and chronOs) as well as two tricalcium phosphate (TCP) ceramics with either a micron-scale (TCP-B) or submicron-scale (TCP-S) surface structure are characterized and their bone forming potential is evaluated in a canine ectopic implantation model. After 12 weeks of implantation in the paraspinal muscle of four beagles, sporadic bone (0.1 ± 0.1%) is observed in two Actifuse implants (2/4), limited bone (2.1 ± 1.4%) in four MBCP implants (4/4) and abundant bone (21.6 ± 4.5%) is formed in all TCP-S implants (4/4). Bone is not observed in any of the Bio-Oss, Bi-Ostetic, Vitoss, chronOs and TCP-B implants (0/4). When correlating the bone forming potential with the physicochemical properties of each material, we observe that the physical characteristics (e.g. grain size and micropore size at the submicron scale) might be the dominant trigger of material directed bone formation via specific mechanotransduction, instead of protein adsorption, surface mineralization and calcium ion release.

Aim: To evaluate the influence of the titanium nitride (TiN) coating on the results of a total knee arthroplasty (TKA)., Methods: A total of 910 patients (338 men; 572 woman), with a mean age of 65 (range 36-94) undergoing 1031 primary TKAs were assessed. Clinical evaluation and patient-reported outcomes were gathered one year after surgery. The questionnaires included the Knee injury and Osteoarthritis Outcome Score (KOOS)-Dutch version, Visual Analogue Scale (VAS) pain scores in rest and during active knee movement, VAS-satisfaction scores, and EQ-5D-3L health scores. This was aimed to assess the overall knee function and patient satisfaction, and to enable us to make a gross comparison to other TKAs., Results: At a mean follow-up of 46 mo (range 1-92) the overall implant survival was 97.7% and 95.1% for any operative reason related to the implant. Twenty-three knees (2.2%) required revision surgery. Arthrofibrosis was the most common indication for a re-operation. The clinical evaluation and patient-reported outcomes revealed good to excellent patient satisfaction and function of the arthroplasty. The median postoperative VAS-pain scores on a scale of 0-100, at one year after surgery were 1 in rest and 2 during movement., Conclusion: The TiN coated, mobile bearing TKA results are excellent and similar to those of other widely used TKA designs. Residual pain of the knee remains a concern and the TiN coating in combination with the mobile bearing does not seem to be the simple solution to this problem. Future research will have to show that the coating gives a better survival than the cobalt chrome version., Competing Interests: Conflict-of-interest statement: No potential conflicts of interest. No financial support.

There is a continuing search for novel synthetic materials as an alternative to autologous bone grafting. Different technologies are explored to promote bone formation, which include the addition of BioGlass™ particles in calcium phosphate (CaP)-based materials and the use of surface modification in the form of submicron surface topographies. In this work, we aimed at comparing the bone formation in a noninstrumented canine interspinous model of moldable formulations of a submicron-surface structured tricalcium phosphate/alkylene oxide copolymer (CaP/AOC) or a tricalcium phosphate/BioGlass/collagen (CaP/BG/C) bone graft material. Intramuscular implantation was carried out as well to evaluate soft tissue responses. Eight mature male mongrel dogs underwent single-level, noninstrumented interspinous implantation, where the bone graft materials were implanted at either side of the spinous processes (L3-L4), with separation by the interspinous ligament ensuring comparison of both materials in each animal (n = 8 per material). The materials were also implanted in paraspinal muscle pouches. Animals were euthanized 12 weeks after surgery and the lumbar spines excised and intramuscular implants retrieved. Undecalcified sections were prepared for histological evaluation and histomorphometry was performed to quantify bone formation and material resorption. After 12 weeks, all submicron structured CaP/AOC implants showed abundant bone formation in the (L3-L4) interspinous space (20.8% ± 6.8%), whereas bone was not found in the CaP/BG/C implants (0% ± 0%). Intramuscularly, the CaP/AOC material triggered significant bone formation (12.0% ± 7.8%), whereas CaP/BG/C did not form any bone. In both the spinal and muscular sites, resorption of the CaP/AOC material was evident by a decrease in Feret diameter of the CaP granules as well as in their histological surface compared with the starting material, whereas CaP/BG/C material had a milder resorption. This study shows that a submicron-surface structured CaP/AOC bone graft material has superior bone-forming properties in both an interspinous implantation model and intramuscularly, as compared with a CaP/BG/C bone graft material.

Surface structure largely affects the inductive bone-forming potential of calcium phosphate (CaP) ceramics in ectopic sites and bone regeneration in critical-sized bone defects. Surface-dependent osteogenic differentiation of bone marrow stromal cells (BMSCs) partially explained the improved bone-forming ability of submicron surface structured CaP ceramics. In this study, we investigated the possible influence of surface structure on different bone-related cells, which may potentially participate in the process of improved bone formation in CaP ceramics. Besides BMSCs, the response of human brain vascular pericytes (HBVP), C2C12 (osteogenic inducible cells), MC3T3-E1 (osteogenic precursors), SV-HFO (pre-osteoblasts), MG63 (osteoblasts) and SAOS-2 (mature osteoblasts) to the surface structure was evaluated in terms of cell proliferation, osteogenic differentiation and gene expression. The cells were cultured on tricalcium phosphate (TCP) ceramics with either micron-scaled surface structure (TCP-B) or submicron-scaled surface structure (TCP-S) for up to 14 days, followed by DNA, alkaline phosphatase (ALP) and quantitative polymerase chain reaction gene assays. HBVP were not sensitive to surface structure with respect to cell proliferation and osteogenic differentiation, but had downregulated angiogenesis-related gene expression (i.e. vascular endothelial growth factor) on TCP-S. Without additional osteogenic inducing factors, submicron-scaled surface structure enhanced ALP activity and osteocalcin gene expression of human (h)BMSCs and C2C12 cells, favoured the proliferation of MC3T3-E1, MG63 and SAOS-2, and increased ALP activity of MC3T3-E1 and SV-HFO. The results herein indicate that cells with osteogenic potency (either osteogenic inducible cells or osteogenic cells) could be sensitive to surface structure and responded to osteoinductive submicron-structured CaP ceramics in cell proliferation, ALP production or osteogenic gene expression, which favour bone regeneration. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

The surface topography of synthetic biomaterials is known to play a role in material-driven osteogenesis. Recent studies show that TGFβ signalling also initiates osteogenic differentiation. TGFβ signalling requires the recruitment of TGFβ receptors (TGFβR) to the primary cilia. In this study, we hypothesize that the surface topography of calcium phosphate ceramics regulates stem cell morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation. We developed a 2D system using two types of tricalcium phosphate (TCP) ceramic discs with identical chemistry. One sample had a surface topography at micron-scale (TCP-B, with a bigger surface structure dimension) whilst the other had a surface topography at submicron scale (TCP-S, with a smaller surface structure dimension). In the absence of osteogenic differentiation factors, human bone marrow stromal cells (hBMSCs) were more spread on TCP-S than on TCP-B with alterations in actin organization and increased primary cilia prevalence and length. The cilia elongation on TCP-S was similar to that observed on glass in the presence of osteogenic media and was followed by recruitment of transforming growth factor-β RII (p-TGFβ RII) to the cilia axoneme. This was associated with enhanced osteogenic differentiation of hBMSCs on TCP-S, as shown by alkaline phosphatase activity and gene expression for key osteogenic markers in the absence of additional osteogenic growth factors. Similarly, in vivo after a 12-week intramuscular implantation in dogs, TCP-S induced bone formation while TCP-B did not. It is most likely that the surface topography of calcium phosphate ceramics regulates primary cilia length and ciliary recruitment of p-TGFβ RII associated with osteogenesis and bone formation. This bioengineering control of osteogenesis via primary cilia modulation may represent a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery applications., Statement of Significance: The surface topography of synthetic biomaterials plays important roles in material-driven osteogenesis. The data presented herein have shown that the surface topography of calcium phosphate ceramics regulates mesenchymal stromal cells (e.g., human bone marrow mesenchymal stromal cells, hBMSCs) with respect to morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation in vitro. Together with bone formation in vivo, our results suggested a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery by the bioengineering control of osteogenesis via primary cilia modulation., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Recently, NOD-SCID IL2Rγ -/- (NSG) mice were implanted with human mesenchymal stromal cells (MSCs) in the presence of ceramic scaffolds or Matrigel to mimic the human bone marrow (BM) microenvironment. This approach allowed the engraftment of leukemic samples that failed to engraft in NSG mice without humanized niches and resulted in a better preservation of leukemic stem cell self-renewal properties. To further improve our humanized niche scaffold model, we genetically engineered human MSCs to secrete human interleukin-3 (IL-3) and thrombopoietin (TPO). In vitro, these IL-3- and TPO-producing MSCs were superior in expanding human cord blood (CB) CD34 + hematopoietic stem/progenitor cells. MLL-AF9-transduced CB CD34 + cells could be transformed efficiently along myeloid or lymphoid lineages on IL-3- and TPO-producing MSCs. In vivo, these genetically engineered MSCs maintained their ability to differentiate into bone, adipocytes, and other stromal components. Upon transplantation of MLL-AF9-transduced CB CD34 + cells, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) developed in engineered scaffolds, in which a significantly higher percentage of myeloid clones was observed in the mouse compartments compared with previous models. Engraftment of primary AML, B-cell ALL, and biphenotypic acute leukemia (BAL) patient samples was also evaluated, and all patient samples could engraft efficiently; the myeloid compartment of the BAL samples was better preserved in the human cytokine scaffold model. In conclusion, we show that we can genetically engineer the ectopic human BM microenvironment in a humanized scaffold xenograft model. This approach will be useful for functional study of the importance of niche factors in normal and malignant human hematopoiesis., (Copyright © 2017 ISEH - International Society for Experimental Hematology. All rights reserved.)

Fabrication of composite scaffolds using stereolithography (SLA) for bone tissue engineering has shown great promises. However, in order to trigger effective bone formation and implant integration, exogenous growth factors are commonly combined to scaffold materials. In this study, we fabricated biodegradable composite scaffolds using SLA and endowed them with osteopromotive properties in the absence of biologics. First we prepared photo-crosslinkable poly(trimethylene carbonate) (PTMC) resins containing 20 and 40wt% of hydroxyapatite (HA) nanoparticles and fabricated scaffolds with controlled macro-architecture. Then, we conducted experiments to investigate how the incorporation of HA in photo-crosslinked PTMC matrices improved human bone marrow stem cells osteogenic differentiation in vitro and kinetic of bone healing in vivo. We observed that bone regeneration was significantly improved using composite scaffolds containing as low as 20wt% of HA, along with difference in terms of osteogenesis and degree of implant osseointegration. Further investigations revealed that SLA process was responsible for the formation of a rich microscale layer of HA corralling scaffolds. To summarize, this work is of substantial importance as it shows how the fabrication of hierarchical biomaterials via surface-enrichment of functional HA nanoparticles in composite polymer stereolithographic structures could impact in vitro and in vivo osteogenesis., Statement of Significance: This study reports for the first time the enhance osteopromotion of composite biomaterials, with controlled macro-architecture and microscale distribution of hydroxyapatite particles, manufactured by stereolithography. In this process, the hydroxyapatite particles are not only embedded into an erodible polymer matrix, as reported so far in the literature, but concentrated at the surface of the structures. This leads to robust in vivo bone formation at low concentration of hydroxyapatite. The reported 3D self-corralling composite architecture provides significant opportunities to develop functional biomaterials for bone repair and tissue engineering., (Copyright © 2017. Published by Elsevier Ltd.)

To begin to understand the mechanisms that regulate self-renewal, differentiation, and transformation of human hematopoietic stem cells or to evaluate the efficacy of novel treatment modalities, stem cells need to be studied in their own species-specific microenvironment. By implanting ceramic scaffolds coated with human mesenchymal stromal cells into immune-deficient mice, we were able to mimic the human bone marrow niche. Thus, we have established a human leukemia xenograft mouse model in which a large cohort of patient samples successfully engrafted, which covered all of the important genetic and risk subgroups. We found that by providing a humanized environment, stem cell self-renewal properties were better maintained as determined by serial transplantation assays and genome-wide transcriptome studies, and less clonal drift was observed as determined by exome sequencing. The human leukemia xenograft mouse models that we have established here will serve as an excellent resource for future studies aimed at exploring novel therapeutic approaches., (© 2016 by The American Society of Hematology.)

Calcium phosphate ceramics with submicron-scaled surface structure can trigger bone formation in non-osseous sites and are expected to enhance bone formation in spine environment. In this study, two tricalcium phosphate ceramics having either a submicron-scaled surface structure (TCP-S) or a micron-scaled one (TCP-B) were prepared and characterized regarding their physicochemical properties. Granules (size 1-2 mm) of both materials were implanted on either left or right side of spinous process, between the two lumbar vertebrae (L3-L4), and in paraspinal muscle of eight beagles. After 12 weeks of implantation, ectopic bone was observed in muscle in TCP-S explants (7.7 ± 3.7%), confirming their ability to inductively form bone in non-osseous sites. In contrast, TCP-B implants did not lead to bone formation in muscle. Abundant bone (34.1 ± 6.6%) was formed within TCP-S implants beside the two spinous processes, while limited bone (5.1 ± 4.5%) was seen in TCP-B. Furthermore, the material resorption of TCP-S was more pronounced than that of TCP-B in both the muscle and spine environments. The results herein indicate that the submicron-scaled surface structured tricalcium phosphate ceramic could enhance bone regeneration as compared to the micron-scaled one in spine environment. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1865-1873, 2016., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Although NOD-SCID IL2Rγ -/- (NSG) xenograft mice are currently the most frequently used model to study human leukemia in vivo, the absence of a human niche severely hampers faithful recapitulation of the disease. We used NSG mice in which ceramic scaffolds seeded with human mesenchymal stromal cells were implanted to generate a human bone marrow (huBM-sc)-like niche. We observed that, in contrast to the murine bone marrow (mBM) niche, the expression of BCR-ABL or MLL-AF9 was sufficient to induce both primary acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). Stemness was preserved within the human niches as demonstrated by serial transplantation assays. Efficient engraftment of AML MLL-AF9 and blast-crisis chronic myeloid leukemia patient cells was also observed, whereby the immature blast-like phenotype was maintained in the huBM-sc niche but to a much lesser extent in mBM niches. We compared transcriptomes of leukemias derived from mBM niches versus leukemias from huBM-like scaffold-based niches, which revealed striking differences in the expression of genes associated with hypoxia, mitochondria and metabolism. Finally, we utilized the huBM-sc MLL-AF9 B-ALL model to evaluate the efficacy of the I-BET151 inhibitor in vivo. In conclusion, we have established human niche models in which the myeloid and lymphoid features of BCR-ABL + and MLL-AF9 + leukemias can be studied in detail.

Strontium (Sr) has been shown to favor bone formation and is used clinically to treat osteoporosis. We have previously reported that Sr addition in apatite/polylactide composites could enhance the BMP-induced bone formation around implants at ectopic site in healthy animals. In this study we aimed to investigate the effectiveness of Sr addition on the local bone formation in osteoporosis. Apatite/polylactide composite granules with different Sr content were loaded with equal amount of rhBMP-2 and implanted intramuscularly in healthy rabbits (Con) and rabbits that received bilateral ovariectomy and daily injection of glucocorticoid (OP) for 12 weeks. The potential effect of Sr on the final volume of BMP-induced bone in both groups was investigated histologically and histomorphometrically. The de novo bone formed in OP implants was significantly less than in Con group when the implants contained no Sr, indicating that the BMP-induced osteogenesis was impaired in OP animals. Sr substitution as low as 0.5 mol% in apatite increased the bone volume in OP implants to levels comparable to that in the Con group, indicating a positive effect of Sr addition on the local bone formation in OP animals. In addition, more adipose tissue formed in parallel with the appearance of cartilage tissue in OP implants, suggesting that the differentiation potential of stem cell in OP animals may have shifted towards adipogenesis and chondrogenesis. From these results, we conclude that the use of Sr addition to enhance the bone growth surrounding implants in osteoporosis merits further study., Statement of Significance: The impaired bone healing capacity of osteoporotic patients might result in poor osteointegration and surgical failure in case implants are placed. In this study we aimed to enhance the bone formation around implants under such scenario by adding strontium as the stimulus. Different from other studies, the samples were loaded with rhBMP-2 and implanted at an ectopic site (spinal muscles of New Zealand rabbits) to exclude the influence of conductive bone repair. The results showed that the addition of strontium could enhance the BMP-2-induced bone formation on implants in osteopenic rabbits to levels comparable to that in healthy rabbits. Secondarily, we observed more adipose tissue and cartilage tissue in osteopenic implants, suggesting the role of adipogenesis and chondrogenesis in osteopenia/osteoporosis., (Copyright © 2015. Published by Elsevier Ltd.)

It has been reported that surface microstructural dimensions can influence the osteoinductivity of calcium phosphates (CaPs), and osteoclasts may play a role in this process. We hypothesised that surface structural dimensions of ≤ 1 μm trigger osteoinduction and osteoclast formation irrespective of macrostructure (e.g., concavities, interconnected macropores, interparticle space) or surface chemistry. To test this, planar discs made of biphasic calcium phosphate (BCP: 80% hydroxyapatite, 20% tricalcium phosphate) were prepared with different surface structural dimensions - either ~ 1 μm (BCP1150) or ~ 2-4 μm (BCP1300) - and no macropores or concavities. A third material was made by sputter coating BCP1150 with titanium (BCP1150Ti), thereby changing its surface chemistry but preserving its surface structure and chemical reactivity. After intramuscular implantation in 5 dogs for 12 weeks, BCP1150 formed ectopic bone in 4 out of 5 samples, BCP1150Ti formed ectopic bone in 3 out of 5 samples, and BCP1300 formed no ectopic bone in any of the 5 samples. In vivo, large multinucleated osteoclast-like cells densely colonised BCP1150, smaller osteoclast-like cells formed on BCP1150Ti, and osteoclast-like cells scarcely formed on BCP1300. In vitro, RAW264.7 cells cultured on the surface of BCP1150 and BCP1150Ti in the presence of osteoclast differentiation factor RANKL (receptor activator for NF-κB ligand) proliferated then differentiated into multinucleated osteoclast-like cells with positive tartrate resistant acid phosphatase (TRAP) activity. However, cell proliferation, fusion, and TRAP activity were all significantly inhibited on BCP1300. These results indicate that of the material parameters tested - namely, surface microstructure, macrostructure, and surface chemistry - microstructural dimensions are critical in promoting osteoclastogenesis and triggering ectopic bone formation.

Material factors such as chemistry, surface microstructure and geometry have shown their influence on osteoinduction of calcium phosphate ceramics. Hereby we report that osteoinduction of a micro-structured biphasic calcium phosphate ceramic (BCP) has a relation with the particle sizes. BCP particles with the size of 212-300 µm, 106-212 µm, 45-106 µm, and smaller than 45 µm were prepared and implanted in paraspinal muscle of dogs for 12 weeks. Histological evaluation of the explants showed abundant bone in all samples with particle size of 212-300 µm, 106-212 µm, and 45-106 µm, while no bone was seen in any sample having particle size smaller than 45 µm. Bone was formed as early as 3 weeks after implantation in implants having BCP particles bigger than 45 µm and the volume of the formed bone was similar among the implants with particles larger than 45 µm after 12 weeks implantation. The results herein show that a size limitation of microstructured calcium phosphate ceramic particles for osteoinduction. It is most likely that the particle size affect inductive bone formation via macroporous structures for body fluid infiltration, cell/tissue ingrowth and angiogenesis., (© 2014 Wiley Periodicals, Inc.)

The influence of fluoride in poly(d,l-lactide)/apatite composites on ectopic bone formation was evaluated in sheep. Nano-apatite powders with different replacement levels of OH groups by fluoride (F) (0% (F0), 50% (F50), 100% (F100), and excessive (F200)) were co-extruded with poly (d,l-lactide) at a weight ratio of 1:1. Fluoride release from the composites (CF0, CF50, CF100, and CF200) was evaluated in vitro and bone formation was assessed after intramuscular implantation in sheep. After 24 weeks in simulated physiological solution, CF0 and CF50 showed negligible fluoride release, whereas it was considerable from the CF100 and CF200 composites. Histology showed that the incidence of de novo bone formation decreased in implants with increasing fluoride content indicating a negative influence of fluoride on ectopic bone formation. Furthermore, a significant decrease in resorption of the high fluoride-content composites and a reduction in the number of multinucleated giant cells were seen. These results show that instead of promoting, the presence of fluoride in poly(d,l-lactide)/apatite composites seemed to suppresses their resorption and osteoinductive potential in non-osseous sites., (© 2014 Wiley Periodicals, Inc.)

The presence of micropores in calcium phosphate (CaP) ceramics has shown its important role in initiating inductive bone formation in ectopic sites. To investigate how microporous CaP ceramics trigger osteoinduction, we optimized two biphasic CaP ceramics (i.e., BCP-R and BCP-S) to have the same chemical composition, equivalent surface area per volume, comparable protein adsorption, similar ion (i.e., calcium and phosphate) exchange and the same surface mineralization potential, but different surface architecture. In particular, BCP-R had a surface roughness (Ra) of 325.4 ± 58.9 nm while for BCP-S it was 231.6 ± 35.7 nm. Ceramic blocks with crossing or noncrossing channels of 250, 500, 1000, and 2000 µm were implanted in paraspinal muscle of dogs for 12 weeks. The percentage of bone volume in the channels was not affected by the type of pores (i.e., crossing vs. closed) or their size, but it was greatly influenced by the ceramic type (i.e., BCP-R vs. BCP-S). Significantly, more bone was formed in the channels of BCP-R than in those of BCP-S. Since the two CaP ceramics differed only in their surface architecture, the results hereby demonstrate that microporous CaP ceramics may induce ectopic osteogenesis through surface architecture., (© 2014 Wiley Periodicals, Inc.)

Composite materials of photo-crosslinked poly(trimethylene carbonate) and nanoscale hydroxyapatite were prepared and their mechanical characteristics for application as orbital floor implants were assessed. The composites were prepared by solvent casting poly(trimethylene carbonate) macromers with varying amounts of nano-hydroxyapatite and subsequent photo-crosslinking. The incorporation of the nano-hydroxyapatite into the composites was examined by thermogravimetric analysis, scanning electron microscopy and gel content measurements. The mechanical properties were investigated by tensile testing and trouser tearing experiments. Our results show that nano-hydroxyapatite particles can readily be incorporated into photo-crosslinked poly(trimethylene carbonate) networks. Compared to the networks without nano-hydroxyapatite, incorporation of 36.3 wt.% of the apatite resulted in an increase of the E modulus, yield strength and tensile strength from 2.2 MPa to 51 MPa, 0.5 to 1.4 N/mm2 and from 1.3 to 3.9 N/mm2, respectively. We found that composites containing 12.4 wt.% nano-hydroxyapatite had the highest values of strain at break, toughness and average tear propagation strength (376% , 777 N/mm2 and 3.1 N/mm2, respectively).

A resorbable bone graft substitute should mimic native bone in its capacity to support bone formation and be remodeled by osteoclasts (OCl) or other multinucleated cells such as foreign body giant cells (FBGC). We hypothesize that by changing the scale of surface architecture of beta-tricalcium phosphate (TCP), cellular resorption can be influenced. CD14(+) monocyte precursors were isolated from human peripheral blood (n = 4 independent donors) and differentiated into OCl or FBGC on the surface of TCP discs comprising either submicron- or micron-scale surface topographical features (TCPs and TCPb, respectively). On submicrostructured TCPs, OCl survived, fused, differentiated, and extensively resorbed the substrate; however, on microstructured TCPb, OCl survival, TRAP activation, and fusion were attenuated. Importantly, no resorption was observed on microstructured TCPb. By confocal microscopy, OCl formed on TCPs contained numerous actin rings allowing for resorption, but not on TCPb. In comparison, FBGC could not resorb either TCP material, suggesting that osteoclast-specific machinery is necessary to resorb TCP. By tuning surface architecture, it appears possible to control osteoclast resorption of calcium phosphate. This approach presents a useful strategy in the design of resorbable bone graft substitutes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The microporosity of calcium phosphate (CaP) ceramics has been shown to have an essential role in osteoinduction by CaP ceramics after ectopic implantation. Here we show that it is not the microporosity but the size of surface microstructural features that is the most likely osteogenic factor. Two tricalcium phosphate (TCP) ceramics, namely TCP-S and TCP-B, were fabricated with equivalent chemistry and similar microporosity but different sizes of surface microstructural features. TCP-S has a grain size of 0.99 ± 0.20 μm and a micropore size of 0.65 ± 0.25 μm, while TCP-B displays a grain size of 3.08 ± 0.52 μm and a micropore size of 1.58 ± 0.65 μm. In vitro, both cell proliferation and osteogenic differentiation were significantly enhanced when human bone marrow stromal cells were cultured on TCP-S without any osteogenic growth factors, compared to TCP-B ceramic granules. The possible involvement of direct contact between cells and the TCP ceramic surface in osteogenic differentiation is also shown with a trans-well culture model. When the ceramic granules were implanted in paraspinal muscle of dogs for 12 weeks, abundant bone was formed in TCP-S (21 ± 10% bone in the available space), whereas no bone was formed in any of the TCP-B implants. The current in vitro and in vivo data reveal that the readily controllable cue, i.e. the size of the surface microstructure, could be sufficient to induce osteogenic differentiation of mesenchymal stem cells, ultimately leading to ectopic bone formation in calcium phosphate ceramics., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Bone graft substitutes such as calcium phosphates are subject to the innate inflammatory reaction, which may bear important consequences for bone regeneration. We speculate that the surface architecture of osteoinductive β-tricalcium phosphate (TCP) stimulates the differentiation of invading monocyte/macrophages into osteoclasts, and that these cells may be essential to ectopic bone formation. To test this, porous TCP cubes with either submicron-scale surface architecture known to induce ectopic bone formation (TCPs, positive control) or micron-scale, non-osteoinductive surface architecture (TCPb, negative control) were subcutaneously implanted on the backs of FVB strain mice for 12 weeks. Additional TCPs samples received local, weekly injections of liposome-encapsulated clodronate (TCPs + LipClod) to deplete invading monocyte/macrophages. TCPs induced osteoclast formation, evident by positive tartrate resistant acid phosphatase (TRAP) cytochemical staining and negative macrophage membrane marker F4/80 immunostaining. No TRAP positive cells were found in TCPb or TCPs + LipClod, only F4/80 positive macrophages and foreign body giant cells. TCPs stimulated subcutaneous bone formation in all implants, while no bone could be found in TCPb or TCPs + LipClod. In agreement, expression of bone and osteoclast gene markers was upregulated in TCPs versus both TCPb and TCPs + LipClod, which were equivalent. In summary, submicron-scale surface structure of TCP induced osteoclastogenesis and ectopic bone formation in a process that is blocked by monocyte/macrophage depletion., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A current challenge of synthetic bone graft substitute design is to induce bone formation at a similar rate to its biological resorption, matching bone's intrinsic osteoinductivity and capacity for remodelling. We hypothesise that both osteoinduction and resorption can be achieved by altering surface microstructure of beta-tricalcium phosphate (TCP). To test this, two TCP ceramics are engineered with equivalent chemistry and macrostructure but with either submicron- or micron-scale surface architecture. In vitro, submicron-scale surface architecture differentiates larger, more active osteoclasts--a cell type shown to be important for both TCP resorption and osteogenesis--and enhances their secretion of osteogenic factors to induce osteoblast differentiation of human mesenchymal stem cells. In an intramuscular model, submicrostructured TCP forms 20 % bone in the free space, is resorbed by 24 %, and is densely populated by multinucleated osteoclast-like cells after 12 weeks; however, TCP with micron-scale surface architecture forms no bone, is essentially not resorbed, and contains scarce osteoclast-like cells. Thus, a novel submicron-structured TCP induces substantial bone formation and is resorbed at an equivalent rate, potentially through the control of osteoclast-like cells.

In the treatment of orbital floor fractures, bone is ideally regenerated. The materials currently used for orbital floor reconstruction do not lead to the regeneration of bone. Our objective was to render polymeric materials based on poly(trimethylene carbonate) (PTMC) osteoinductive, and to evaluate their suitability for use in orbital floor reconstruction. For this purpose, osteoinductive biphasic calcium phosphate (BCP) particles were introduced into a polymeric PTMC matrix. Composite sheets containing 50 wt% BCP particles were prepared. Also laminates with poly(D,L-lactide) (PDLLA) were prepared by compression moulding PDLLA films onto the composite sheets. After sterilisation by gamma irradiation, the sheets were used to reconstruct surgically-created orbital floor defects in sheep. The bone inducing potential of the different implants was assessed upon intramuscular implantation. The performance of the implants in orbital floor reconstruction was assessed by cone beam computed tomography (CBCT). Histological evaluation revealed that in the orbital and intramuscular implantations of BCP containing specimens, bone formation could be seen after 3 and 9 months. Analysis of the CBCT scans showed that the composite PTMC sheets and the laminated composite sheets performed well in orbital floor reconstruction. It is concluded that PTMC/BCP composites and PTMC/BCP composites laminated with PDLLA have osteoinductive properties and seem suitable for use in orbital floor reconstruction.

Synthetic bone graft substitutes based on composites consisting of a polymer and a calcium-phosphate (CaP) ceramic are developed with the aim to satisfy both mechanical and bioactivity requirements for successful bone regeneration. In the present study, we have employed extrusion to produce a composite consisting of 50 wt.% poly(D,L-lactic acid) (PLA) and 50 wt.% nano-sized hydroxyapatite (HA) powder, achieving homogeneous distribution of the ceramic within the polymeric phase. In vitro, in both a simulated physiological saline (SPS) and a simulated body fluid (SBF), a greater weight loss was observed for PLA/HA than for PLA particles upon 12-week immersion. Furthermore, in SPS, a continuous release of calcium and phosphate from the composite was measured, whereas in SBF, decrease of the amount of the two ions in the solution was observed both for PLA and PLA/HA accompanied with the formation of a CaP layer on the surface. In vitro characterization of the composite bioactivity was performed by culturing human mesenchymal stromal cells (hMSCs) and assessing proliferation and osteogenic differentiation, with PLA as a control. Both PLA/HA composite and PLA control were shown to support hMSCs proliferation over a period of two weeks. In addition, the composite significantly enhanced alkaline phosphatase (ALP) activity of hMSCs in osteogenic medium as compared with the polymer control. A novel implant design was employed to develop implants from dense, extruded materials, suitable for testing osteoinductivity in vivo. In a preliminary study in dogs, PLA/HA composite implants induced heterotopic bone formation upon 12-week intramuscular implantation in all animals, in contrast to PLA control, which was not osteoinductive. Unlike in vitro, a more pronounced degradation of PLA was observed in vivo as compared with PLA/HA composite.

Zinc-containing tricalcium phosphate (Zn-TCP) was synthesized to investigate the role of zinc in osteoblastogenesis, osteoclastogenesis and in vivo bone induction in an ectopic implantation model. Zinc ions were readily released in the culture medium. Zn-TCP with the highest zinc content enhanced the alkaline phosphatase activity of human bone marrow stromal cells and tartrate-resistant acid phosphatase activity, as well as multinuclear giant cell formation of RAW264.7 monocyte/macrophages. RAW264.7 cultured with different dosages of zinc supplements in medium with or without zinc-free TCP showed that zinc could influence both the activity and the formation of multinuclear giant cells. After a 12-week implantation in the paraspinal muscle of canines, de novo bone formation and bone incidence increased with increasing zinc content in Zn-TCP - up to 52% bone in the free space. However, TCP without zinc induced no bone formation. Although the observed bone induction cannot be attributed to zinc release alone, these results indicate that zinc incorporated in TCP can modulate bone metabolism and render TCP osteoinductive, indicating to a novel way to enhance the functionality of this synthetic bone graft material., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

In bone tissue regeneration, certain polymer and calcium-phosphate-based composites have been reported to enhance some biological surface phenomena, facilitating osteoinduction. Although the crucial role of inorganic fillers in heterotopic bone formation by such materials has been shown, no reports have been published on the potential effects the polymer phase may have. The present work starts from the assumption that the polymer molecular weight regulates the fluid uptake, which determines the hydrolysis rate and the occurrence of biological surface processes. Here, two composites were prepared by extruding two different molecular weight L/D,L-lactide copolymers with calcium phosphate apatite. The lower molecular weight copolymer allowed larger fluid uptake in the composite thereof, which was correlated with a higher capacity to adsorb proteins in vitro. Further, the large fluid absorption led to a quicker composite degradation that generated rougher surfaces and enhanced ion release. Following intramuscular implantation in sheep, only the composite with the lower molecular weight polymer could induce heterotopic bone formation. Besides influencing the biological potential of composites, the molecular weight also regulated their viscoelastic behaviour under cyclic stresses. The results lead to the conclusion that designing biomaterials with appropriate physico-chemical characteristics is crucial for bone tissue regeneration in mechanical load-bearing sites., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The most common application of bone grafts is spinal fusion surgery, in which the use of iliac crest autograft is the gold standard. Harvesting of autograft, however, requires an extra surgical procedure, which is associated with additional morbidity. Allograft is the well-known alternative, but it is generally considered less effective in posterior fusions. Therefore, the need for an effective alternative remains. Recently, it was shown that ceramics can be endowed with biologically instructive properties by changing the basic parameters of the material. In this study, we compared a novel tricalcium phosphate ceramic (TCP) to iliac crest autograft and allograft, in instrumented posterolateral fusions in a goat model. A total of nine goats were included, who underwent a two-level lumbar fusion. Each side of the spine was randomized into one type of graft: iliac crest autograft; fresh-frozen allograft; TCP alone; or TCP combined with local autograft (50:50). The fusion rates after 16 weeks were comparable between the groups (autograft 3/8, allograft 4/8, TCP 4/8, and TCP/local autograft 5/8). Calculation of the fusion volume on computed tomography images, showed significantly greater volume in the control groups (autograft 7.8 mL and allograft 8.9 mL) compared with the groups with TCP (TCP 6.1 mL and TCP/local autograft 6.0 mL). No adverse tissue response was seen on histological analysis and TCP was almost completely resorbed. The results demonstrate that TCP is capable of achieving fusion at a similar rate to iliac crest autograft in posterolateral fusions, while almost completely resorbing within 16 weeks. Despite the lower fusion volume, the TCP is a promising alternative circumventing the disadvantages of autograft and allograft.

Bone tissue is a dynamic composite system that adapts itself, in response to the surrounding daily (cyclic) mechanical stimuli, through an equilibrium between growth and resorption processes. When there is need of synthetic bone grafts, the biggest issue is to support bone regeneration without causing mechanically-induced bone resorption. Apart from biological properties, such degradable materials should initially support and later leave room to bone formation. Further, dynamic mechanical properties comparable to those of bone are required. In this study we prepared composites comprising calcium phosphate and L-lactide/D-lactide copolymer in various content ratios using the extrusion method. We evaluated the effect of the inorganic filler amount on the polymer phase (i.e. on the post-extrusion intrinsic viscosity). We then studied their in vitro degradation and dynamic mechanical properties (in dry and humid conditions). By increasing the filler content, we observed significant decrease of the intrinsic viscosity of the polymer phase during the extrusion process. Composites containing higher amounts of apatite had faster degradation, and were also mechanically stiffer. But, due to the lower intrinsic viscosity of their polymer phase, they had larger damping properties. Besides this, higher amounts of apatite also rendered the composites more hydrophilic letting them absorb more water and causing them the largest decrease in stiffness. These results show the importance of filler content in controlling the properties of such composites. Further, in this study we observed that the viscoelastic properties of the composite containing 50wt% apatite were comparable to those of dry human cortical bone., (Copyright © 2013 Elsevier Ltd. All rights reserved.)