Demineralized bone matrix and human cancellous bone enhance fixation of porous‐coated titanium implants in sheep

Allogenic bone graft has been considered the gold standard in connection with bone graft material in revision joint arthroplasty. However, the lack of osteogenic potential and the risk of disease transmission are clinical challenges. The use of osteoinductive materials, such as demineralized bone matrix (DBM), alone or in combination with allograft or commercially available human cancellous bone (CB), may replace allografts, as they have the capability of inducing new bone and improving implant fixation through enhancing bone ongrowth. The purpose of this study was to investigate the effect of DBM alone, DBM with CB, or allograft on the fixation of porous‐coated titanium implants. DBM100 and CB produced from human tissue were included. Both materials are commercially available. DBM granules are placed in pure DBM and do not contain any other carrier. Titanium alloy implants, 10 mm long × 10 mm diameter, were inserted bilaterally into the femoral condyles of eight skeletally mature sheep. Thus, four implants with a concentric gap of 2 mm were implanted in each sheep. The gap was filled with: (a) DBM; (b) DBM:CB at a ratio of 1:3; (c) DBM:allograft at a ratio of 1:3; or (d) allograft (gold standard), respectively. A standardized surgical procedure was used. At sacrifice 6 weeks after implantation, both distal femurs were harvested. The implant fixation was evaluated by mechanical push‐out testing to test shear mechanical properties between implant and the host bone and by histomorphometry. Non‐parametric tests were applied; p < 0.05 was considered significant. Mechanical fixation showed that the strengths among the DBM/CB, DBM/allograft and allograft groups were not statistically different. The strength of the DBM group was 0.01 MPa, which was statistical significantly lower than the other three groups (p < 0.05). Histomorphometry results showed that the bone ongrowth in the DBM group was statistically significantly lower than the other three groups, while the volume fraction of new bone showed no significant difference among all the groups. Our data revealed that adding DBM to CB or to allograft resulted in comparable mechanical properties relative to the gold standard, allograft. We found inferior early effects of DBM alone on the fixation of porous‐coated titanium implant in this animal model, while the long‐term effects have to be investigated. The combination of DBM with CB, which can be used off the shelf, may represent an alternative to allograft. A cost–benefit analysis is necessary before application in clinical trial. Copyright © 2013 John Wiley & Sons, Ltd.     

Engineering axially vascularized bone in the sheep arteriovenous‐loop model

Treatment of complex bone defects in which vascular supply is insufficient is still a challenge. To overcome the limitations from autologous grafts, a sheep model has been established recently, which is characterized by the development of an independent axial vascularization of a bioartificial construct, permitting microsurgical transplantation. To engineer independently axially vascularized bone tissue in the sheep arteriovenous (AV)‐loop model, mesenchymal stem cells (MSCs), without and in combination with recombinant human bone morphogenetic protein‐2 (rhBMP‐2), were harvested and directly autotransplanted in combination with β‐tricalcium phosphate–hydroxyapatite (β‐TCP–HA) granules into sheep in this study. After explantation after 12 weeks, histological and immunohistochemical evaluation revealed newly formed bone in both groups. An increased amount of bone area was obtained using directly autotransplanted MSCs with rhBMP‐2 stimulation. Osteoblastic and osteoclastic cells were detected adjacent to the newly formed bone, revealing an active bone remodelling process. Directly autotransplanted MSCs can be found close to the β‐TCP–HA granules and are contributing to bone formation. Over time, magnetic resonance imaging (MRI) and micro‐computed tomography (μCT) imaging confirmed the dense vascularization arising from the AV‐loop. This study shows de novo engineering of independently axially vascularized transplantable bone tissue in clinically significant amounts, using directly autotransplanted MSCs and rhBMP‐2 stimulation in about 12 weeks in the sheep AV‐loop model. This strategy of engineering vascularized transplantable bone tissue could be possibly transferred to the clinic in the future in order to augment current reconstructive strategies. Copyright © 2012 John Wiley & Sons, Ltd.     

BMP‐2‐transduced human bone marrow stem cells enhance neo‐bone formation in a rat critical‐sized femur defect

Synthetic graft materials are considered as possible substitutes for cancellous bone, but lack osteogenic and osteoinductive properties. In this study, we investigated how composite scaffolds of βTCP containing osteogenic human bone marrow mesenchymal stem cells (hBMSCs) and osteoinductive bone morphogenetic protein‐2 (BMP‐2) influenced the process of fracture healing. hBMSCs were loaded into βTCP scaffolds 24 h before implantation in a rat critical‐sized bone defect. hBMSCs were either stimulated with rhBMP‐2 or transduced with BMP‐2 by gene transfer. The effect of both protein stimulation and gene transfer was compared for osteogenic outcome. X‐rays were conducted at weeks 0, 1, 3, 6, 9 and 12 post‐operatively. In addition, bone‐labelling fluorochromes were applied at 0, 3, 6 and 9 weeks. Histological analysis was performed for the amount of callus tissue and cartilage formation. At 6 weeks, the critical‐sized defect in 33% of the rats treated with the Ad‐BMP‐2‐transduced hBMSCs/βTCP scaffolds was radiographically bridged. In contrast, in only 10% of the rats treated with rhBMP2/hBMSCs, 12 weeks post‐treatment, the bone defect was closed in all treated rats of the Ad‐BMP‐2 group except for one. Histology showed significantly higher amounts of callus formation in both Ad‐BMP‐2‐ and rhBMP‐2‐treated rats. The amount of neocartilage was less pronounced in both BMP‐2‐related groups. In summary, scaffolds with BMP‐2‐transduced hBMSCs performed better than those with the rhBMP2/hBMSCs protein. These results suggest that combinations of osteoconductive biomaterials with genetically modified MSCs capable of secreting osteoinductive proteins may represent a promising alternative for bone regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro and in vivo investigation of bisphosphonate‐loaded hydroxyapatite particles for peri‐implant bone augmentation

Locally applied bisphosphonates, such as zoledronate, have been shown in several studies to inhibit peri‐implant bone resorption and recently to enhance peri‐implant bone formation. Studies have also demonstrated positive effects of hydroxyapatite (HA) particles on peri‐implant bone regeneration and an enhancement of the anti‐resorptive effect of bisphosphonates in the presence of calcium. In the present study, both hydroxyapatite nanoparticles (nHA) and zoledronate were combined to achieve a strong reinforcing effect on peri‐implant bone. The nHA–zoledronate combination was first investigated in vitro with a pre‐osteoclastic cell assay (RAW 264.7) and then in vivo in a rat model of postmenopausal osteoporosis. The in vitro study confirmed that the inhibitory effect of zoledronate on murine osteoclast precursor cells was enhanced by loading the drug on nHA. For the in vivo investigation, either zoledronate‐loaded or pure nHA were integrated in hyaluronic acid hydrogel. The gels were injected in screw holes that had been predrilled in rat femoral condyles before the insertion of miniature screws. Micro‐CT‐based dynamic histomorphometry and histology revealed an unexpected rapid mineralization of the hydrogel in vivo through formation of granules, which served as scaffold for new bone formation. The delivery of zoledronate‐loaded nHA further inhibited a degradation of the mineralized hydrogel as well as a resorption of the peri‐implant bone as effectively as unbound zoledronate. Hyaluronic acid with zoledronate‐loaded nHA, thanks to its dual effect on inducing a rapid mineralization and preventing resorption, is a promising versatile material for bone repair and augmentation. Copyright © 2015 John Wiley & Sons, Ltd.     

Gas‐foamed poly(lactide‐co‐glycolide) and poly(lactide‐co‐glycolide) with bioactive glass fibres demonstrate insufficient bone repair in lapine osteochondral defects

Deep osteochondral defects may leave voids in the subchondral bone, increasing the risk of joint structure collapse. To ensure a stable foundation for the cartilage repair, bone grafts can be used for filling these defects. Poly(lactide‐co‐glycolide) (PLGA) is a biodegradable material that improves bone healing and supports bone matrix deposition. We compared the reparative capacity of two investigative macroporous PLGA‐based biomaterials with two commercially available bone graft substitutes in the bony part of an intra‐articular bone defect created in the lapine femur. New Zealand white rabbits (n = 40) were randomized into five groups. The defects, 4 mm in diameter and 8 mm deep, were filled with neat PLGA; a composite material combining PLGA and bioactive glass fibres (PLGA–BGf); commercial beta‐tricalcium phosphate (β‐TCP) granules; or commercial bioactive glass (BG) granules. The fifth group was left untreated for spontaneous repair. After three months, the repair tissue was evaluated with X‐ray microtomography and histology. Relative values comparing the operated knee with its contralateral control were calculated. The relative bone volume fraction (?BV/TV) was largest in the β‐TCP group (p ≤ 0.012), which also showed the most abundant osteoid. BG resulted in improved bone formation, whereas defects in the PLGA–BGf group were filled with fibrous tissue. Repair with PLGA did not differ from spontaneous repair. The PLGA, PLGA–BGf, and spontaneous groups showed thicker and sparser trabeculae than the commercial controls. We conclude that bone repair with β‐TCP and BG granules was satisfactory, whereas the investigational PLGA‐based materials were only as good as or worse than spontaneous repair.     

Spatial distribution and survival of human and goat mesenchymal stromal cells on hydroxyapatite and β‐tricalcium phosphate

The combination of scaffolds and mesenchymal stromal cells (MSCs) is a promising approach in bone tissue engineering (BTE). Knowledge on the survival, outgrowth and bone‐forming capacity of MSCs in vivo is limited. Bioluminescence imaging (BLI), histomorphometry and immunohistochemistry were combined to study the fate of gene‐marked goat and human MSCs (gMSCs, hMSCs) on scaffolds with different osteoinductive properties. Luciferase–GFP‐labelled MSCs were seeded on hydroxyapatite (HA) or β‐tricalcium phosphate (TCP), cultured for 7 days in vitro in osteogenic medium, implanted subcutaneously in immunodeficient mice and monitored with BLI for 6 weeks. The constructs were retrieved and processed for histomorphometry and detection of luciferase‐positive cells (LPCs). For gMSCs, BLI revealed doubling of signal after 1 week, declining to 60% of input after 3 weeks and remaining constant until week 6. hMSCs showed a constant decrease of BLI signal to 25% of input, indicating no further expansion. Bone formation of gMSCs was two‐fold higher on TCP than HA. hMSCs and gMSCs control samples produced equal amounts of bone on TCP. Upon transduction, there was a four‐fold reduction in bone formation compared with untransduced hMSCs, and no bone was formed on HA. LPCs were detected at day 14, but were much less frequent at day 42. Striking differences were observed in spatial distribution. MSCs in TCP were found to be aligned and interconnected on the surface but were scattered in an unstructured fashion in HA. In conclusion, the spatial distribution of MSCs on the scaffold is critical for cell–scaffold‐based BTE. Copyright © 2012 John Wiley & Sons, Ltd.     

Bone regeneration by means of a three‐dimensional printed scaffold in a rat cranial defect

Recently, computer‐designed three‐dimensional (3D) printing techniques have emerged as an active research area with almost unlimited possibilities. In this study, we used a computer‐designed 3D scaffold to drive new bone formation in a bone defect. Poly‐L‐lactide (PLLA) and bioactive β‐tricalcium phosphate (TCP) were simply mixed to prepare ink. PLLA + TCP showed good printability from the micronozzle and solidification within few seconds, indicating that it was indeed printable ink for layer‐by‐layer printing. In the images, TCP on the surface of (and/or inside) PLLA in the printed PLLA + TCP scaffold looked dispersed. MG‐63 cells (human osteoblastoma) adhered to and proliferated well on the printed PLLA + TCP scaffold. To assess new bone formation in vivo, the printed PLLA + TCP scaffold was implanted into a full‐thickness cranial bone defect in rats. The new bone formation was monitored by microcomputed tomography and histological analysis of the in vivo PLLA + TCP scaffold with or without MG‐63 cells. The bone defect was gradually spontaneously replaced with new bone tissues when we used both bioactive TCP and MG‐63 cells in the PLLA scaffold. Bone formation driven by the PLLA + TCP30 scaffold with MG‐63 cells was significantly greater than that in other experimental groups. Furthermore, the PLLA + TCP scaffold gradually degraded and matched well the extent of the gradual new bone formation on microcomputed tomography. In conclusion, the printed PLLA + TCP scaffold effectively supports new bone formation in a cranial bone defect.     

羟基磷灰石/外消旋聚乳酸复合材料生物活性和生物相容性的体内实验

背景：外消旋聚乳酸在体内最主要的降解机制是水解,其与羟基磷灰石复合后,复合材料的生物降解率及失重率是否会有所改善。 目的：观察羟基磷灰石/外消旋聚乳酸复合材料体内植入兔股骨缺损后随时间延长界面及结构的变化。 设计：随机分组对照观察。 单位：武汉理工大学生物材料与工程研究中心。 材料：选用40只健康成年日本大耳白兔,体质量2.0～2.5kg,雌雄各半,由湖北省动物实验中心提供[合格证号：SCXK（鄂）2003-0005]。 方法：实验于2005-06/2006-03在湖北生物材料工程技术研究中心完成。①随机摸球法将大耳白兔分成2组：羟基磷灰石/外消旋聚乳酸组和外消旋聚乳酸对照组,每组20只。各组实验兔麻醉后,于兔左前肢股骨髁部分别将羟基磷灰石/外消旋聚乳酸及外消旋聚乳酸圆柱体植入骨腔洞内,圆柱体可略高于骨质表面。用骨膜覆盖样本材料,复位皮肤及骨膜组织瓣。②实验评估：两组实验兔于造模后3,6,12及24周将植入材料及周围骨组织完整取出,使用扫描电子显微镜进行观察各材料内植入后随时间延长界面及结构的变化。主要观察指标：两组材料内植入后随时间延长界面及结构的变化。 结果：纳入实验兔40只均进入结果分析。材料植入体内后,羟基磷灰石颗粒从材料表面脱落,成纤维细胞向组织内长入,并伴有少量新生骨痂的形成,显示羟基磷灰石/外消旋聚乳酸复合材料具有一定的成骨性和骨连接性。在植入24周时界面观察显示,材料被组织分隔包裹,新生骨组织长入材料,骨愈合情况良好,显示羟基磷灰石/外消旋聚乳酸材料具有良好的生物相容性。对于可生物降解的外消旋聚乳酸聚合物,体内水解是最主要的降解机制,其降解速率由于与羟基磷灰石材料的复合而减小。羟基磷灰石/外消旋聚乳酸复合材料具有成骨和骨连接能力。 结论：羟基磷灰石/外消旋聚乳酸复合材料具有成骨性和骨连接性;由于复合材料降解速率减小而相应改善了其生物相容性;羟基磷灰石/外消旋聚乳酸复合材料适合临床应用于可吸收内固定材料。     

Enhanced bone regeneration by gelatin–β‐tricalcium phosphate composites enabling controlled release of bFGF

The objective of this study was to investigate the feasibility of biodegradable gelatin–β‐tricalcium phosphate (β‐TCP) composites as a cell scaffold and controlled‐release carrier of basic fibroblast growth factor (bFGF) suitable for inducing bone regeneration at a segmental bone defect. The composite of gelatin sponge and β‐TCP granules had an interconnected pore structure with an average size of 340 µm. The composite provided the controlled release of bFGF over 2 weeks. Segmental, critical‐sized, bone defects of 20 mm length were created in the ulnas of New Zealand white rabbits and the gelatin–β‐TCP composites, with or without incorporated bFGF, were implanted into the defects. Bone regeneration and β‐TCP resorption profiles were evaluated by microcomputed tomography scanner analysis and haematoxylin and eosin staining. The composites incorporating bFGF promoted significantly higher bone regeneration at the defect site as compared to the bFGF‐free composites. The controlled release of biologically active bFGF from the composites may possibly be achieved through the biodegradation of the composites, resulting in the promotion of bone regeneration. We conclude that the biodegradable gelatin–β‐TCP composite is a promising scaffold for bone regeneration that enables the controlled release of bFGF. Copyright © 2012 John Wiley & Sons, Ltd.     

Osteogenesis of adipose‐derived stem cells on polycaprolactone–β‐tricalcium phosphate scaffold fabricated via selective laser sintering and surface coating with collagen type I

The current study aimed to fabricate three‐dimensional (3D) polycaprolactone (PCL), polycaprolactone and β‐tricalcium phosphate (PCL–TCP) scaffolds via a selective laser‐sintering technique (SLS). Collagen type I was further coated onto PCL–TCP scaffolds to form PCL–TCP–COL scaffolds. The physical characters of these three scaffolds were analysed. The osteogenic potential of porcine adipose‐derived stem cells (pASCs) was compared among these three scaffolds in order to find an optimal scaffold for bone tissue engineering. The experimental results showed no significant differences in pore size and porosity among the three scaffolds; the porosity was ca. 75–77% and the pore size was ca. 300–500 µm in all three. The compressive modulus was increased from 6.77 ± 0.19 to 13.66 ± 0.19 MPa by adding 30% β‐TCP into a 70% PCL scaffold. No significant increase of mechanical strength was found by surface‐coating with collagen type I. Hydrophilicity and swelling ratios showed statistical elevation (p < 0.05) after collagen type I was coated onto the PCL–TCP scaffolds. The in vitro study demonstrated that pASCs had the best osteogenic differentiation on PCL–TCP–COL group scaffolds, due to the highest ALP activity, osteocalcin mRNA expression and mineralization. A nude mice experiment showed better woven bone and vascular tissue formation in the PCL–TCP–COL group than in the PCL group. In conclusion, the study demonstrated the ability to fabricate 3D, porous PCL–TCP composite scaffolds (PCL:TCP = 70:30 by weight) via an in‐house‐built SLS technique. In addition, the osteogenic ability of pASCs was found to be enhanced by coating COL onto the PCL–TCP scaffolds, both in vitro and in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

The effects of different vascular carrier patterns on the angiogenesis and osteogenesis of BMSC–TCP‐based tissue‐engineered bone in beagle dogs

Bone repair using tissue‐engineered bone (TEB) in a large defect or accompanied by a poor recipient vascular bed is a long‐standing challenge. Surgical vascular carrier patterns of vascular bundle (VB) and arteriovenous loop (AV loop) have been shown to improve the vascularization and repair capacity of TEB. However, the effects of these different vascular carrier patterns on angiogenesis and osteogenesis in TEB have never been evaluated. Here, TEB was constructed with bone marrow mesenchymal stem cells (BMSCs) and β‐TCP and prevascularized by the VB or AV loop technique in beagle dogs. The vascularization and bone formation in TEB were quantitatively compared using Microfil perfusion, histological examination and CT and micro‐CT analyses. The distribution and constitution of the neovasculature were analysed to determine the underlying mechanism of angiogenesis. The results showed that prevascularized TEB generated bone tissue faster and more homogeneously than untreated TEB. The VB technique was found to strike a better balance between bone regeneration and β‐TCP scaffold degradation than the AV loop strategy, which resulted in more vascularization but less bone yield, due to faster degradation of the β‐TCP scaffold. This study indicates that a suitable triangular relationship, composed of bone regeneration, scaffold degradation and vasculature, is critical to TEB construction. Copyright © 2015 John Wiley & Sons, Ltd.     

Segmental composite porous scaffolds with either osteogenesis or anti‐bone resorption properties tested in a rabbit ulna defect model

A functional biomaterial with a therapeutic effect is desirable as an adjuvant therapy to enhance bone formation and prevent local recurrence of bone tumours, especially when the resection margins are not identifiable. In this study, novel composite materials were developed with dual properties of osteopromotion and bone resorption to mimic the tumour inhibition effect, including water‐soluble phosphorylated chitosan (P‐chitosan) for increasing osteoblasts activity and disodium (1 → 4)‐2‐deoxy‐2‐sulphoamino‐β‐d ‐glucopyranuronan (S‐chitosan) for inhibiting bone resorption activity. First, P‐chitosan and S‐chitosan were respectively incorporated into two kinds of PLGA/TCP‐based scaffold, i.e. PLGA–TCP–P‐chitosan (P/T/P‐chitosan) and PLGA–TCP–S‐chitosan (P/T/S‐chitosan) scaffolds. We subsequently tested combined scaffolds of PLGA–TCP–P–S–P‐chitosan (P/T/PSP‐chitosan) made of P/T/P‐chitosan and P/T/S‐chitosan to assess their integral effect, on enhancement of bone formation with P/T/P‐chitosan and inhibition of tissue regeneration with P/T/S‐chitosan, in an established rabbit ulnar bone defect model to imitate bone resection post‐bone tumour. To compare bone healing in the defects, the P/T/P‐chitosan group was regarded as a bone formation enhancement group, while the P/T group served as a control. Bone mineral density (BMD) in the P/T/P‐chitosan and P/T/PSP‐chitosan groups were found to be significantly higher than those in the P/T group, while that in the P/T/P‐chitosan group was greater than that in the P/T/PSP‐chitosan group (p < 0.05). These findings demonstrated that P/T/PSP‐chitosan scaffolds possessed more osteogenic potential than the P/T scaffold but less osteogenic effect than the P/T/P‐chitosan scaffold, as the S‐chitosan component inhibited the activities of osteoblasts for bone formation. These findings implied a dual function of the designed P/T/PSP‐chitosan for further preclinical validation and potential applications in the prevention of local recurrence and for enhancing bone repair after bone tumour resection. Copyright © 2013 John Wiley & Sons, Ltd.     

Basic fibroblast growth factor suspended in Matrigel improves titanium implant fixation in ovariectomized rats

Basic fibroblast growth factor (bFGF) has high potential for tissue regeneration; however, its in vivo effects are unpredictable due to the short-term survival. This study sought to evaluate the effects of bFGF suspended in Matrigel on the implant fixation in ovariectomized (OVX) rats. In vitro, the release kinetics of bFGF was tested using an immuno-ligand-assay. In vivo, eighty titanium implants were randomly divided into 4 groups and inserted in the tibiae of forty OVX rats: no treatment group, bFGF alone group, Matrigel alone group and bFGF + Matrigel group. At 3 months after implantation, tibiae were examined by histology, micro-CT and push-out test. We found that Matrigel could prolong the life span of bFGF in vitro with a sustained release during the 21 days. In vivo, bFGF or Matrigel alone had little effect on the fixation of implant in OVX rats, but bFGF suspended in Matrigel induced nearly 2-fold of peri-implant new bone formation and 4-fold of implant mechanical stability when compared to other 3 groups. The results of this study suggest that Matrigel could be used as a carrier of bFGF and prolonged its release around implant, which may improve implant fixation, especially in site of post-menopausal osteoporosis.     

Porous titanium scaffolds with injectable hyaluronic acid–DBM gel for bone substitution in a rat critical‐sized calvarial defect model

Demineralized bone matrix (DBM) is an allograft bone substitute used for bone repair surgery to overcome drawbacks of autologous bone grafting, such as limited supply and donor‐site comorbidities. In view of different demineralization treatments to obtain DBM, we examined the biological performance of two differently demineralized types of DBM, i.e. by acidic treatment using hydrochloric acid (HCl) or treatment with the chelating agent ethylene diamine tetra‐acetate (EDTA). First, we evaluated the osteo‐inductive properties of both DBMs by implanting the materials subcutaneously in rats. Second, we evaluated the effects on bone formation by incorporating DBM in a hyaluronic acid (HA) gel to fill a porous titanium scaffold for use in a critical‐sized calvarial defect model in 36 male Wistar rats. These porous titanium scaffolds were implanted empty or filled with HA gel containing either DBM HCl or DBM EDTA. Ectopically implanted DBM HCl and DBM EDTA did not induce ectopic bone formation over the course of 12 weeks. For the calvarial defects, mean percentages of newly formed bone at 2 weeks were significantly higher for Ti‐Empty compared to Ti–HA + DBM HCl_, but not compared to Ti–HA + DBM EDTA. Significant temporal bone formation was observed for Ti‐Empty and Ti–HA + DBM HCl, but not for Ti–HA + DBM EDTA. At 8 weeks there were no significant differences in values of bone formation between the three experimental constructs. In conclusion, these results showed that, under the current experimental conditions, neither DBM HCl nor DBM EDTA possess osteo‐inductive properties. Additionally, in combination with an HA gel loaded in a porous titanium scaffold, DBM HCl and DBM EDTA showed similar amounts of new bone formation after 8 weeks, which were lower than using the empty porous titanium scaffold. Copyright © 2016 John Wiley & Sons, Ltd.     

Cyclosporine‐impregnated allograft bone sterilized with low‐temperature plasma

Deep‐freezing, freeze‐drying and gamma (γ)‐irradiation have deleterious effects on bone healing and mechanical properties of allograft bones. We tried preparing bone allografts using cyclosporine plus low‐temperature‐plasma sterilization. To explore the feasibility of this method of preparation, segmental defects in the right radii of rabbits were repaired with cyclosporine‐impregnated allograft bones (CABs) sterilized with low‐temperature‐plasma (in the study group) and deep‐frozen/freeze‐dried irradiated allograft bones (D/FIABs) (in the control group). X‐ray and quantitative histological analysis, peripheral blood T lymphocyte subset analysis and CD₂₅ molecule immunohistochemistry stain, the four‐point bending test and safety evaluations were respectively conducted to compare bone‐healing, immunosuppression, mechanical properties and safety between the two groups. X‐ray scores were higher in the study group than those in the control (p = 0.032). There were significant differences in new bone areas at most repairs in both groups (p < 0.05). There were no significant differences in the percentages of CD₄⁺ T, CD₈⁺ T, ratios of CD₄⁺ T:CD₈⁺ T or serum concentrations of GPT/Cr in both groups (p > 0.05). At 16 weeks postoperatively, the density of CD₂₅ molecules in the control group was higher than that in the study group. The ultimate loading in the study group was significantly higher than that in the control (p = 0.048). Bone marrow stromal cells (BMSCs) grew thickly around and on the surface of a cyclosporine‐impregnated allograft. Livers and kidneys in the study and control groups remained histologically normal at 7 days postoperatively. These results indicate that the CAB might be a better material than the D/FIAB in terms of bone healing, preservation of mechanical properties and immunosuppression without severe side‐effects. Copyright © 2010 John Wiley & Sons, Ltd.     

Bone regeneration in minipigs via calcium phosphate cement scaffold delivering autologous bone marrow mesenchymal stem cells and platelet‐rich plasma

Macroporous calcium phosphate cement (CPC) with stem cell seeding is promising for bone regeneration. The objective of this study was to investigate the effects of co‐delivering autologous bone marrow mesenchymal stem cells (BMSCs) and autologous platelet‐rich plasma (PRP) in CPC scaffold for bone regeneration in minipigs for the first time. Twelve female adult Tibet minipigs (12–18 months old) were used. A cylindrical defect with 10 mm height and 8 mm diameter was prepared at the femoral condyle. Two bone defects were created in each minipig, one at each side of the femoral condyle. Three constructs were tested: (1) CPC scaffold (CPC control); (2) CPC seeded with BMSCs (CPC‐BMSC); (3) CPC seeded with BMSCs and PRP (CPC‐BMSC‐PRP). Two time points were tested: 6 and 12 weeks (n = 4). Good integration of implant with surrounding tissues was observed in all groups. At 12 weeks, the CPC‐BMSC‐PRP group had significantly less residual CPC remaining in the defect than the CPC‐BMSC group and the CPC control (p < 0.05). The residual CPC volume for the CPC‐BMSC‐PRP group was half that of the CPC control. New bone formation for CPC‐BMSC‐PRP was more than two‐fold that of the CPC control (p < 0.05). CPC‐BMSC‐PRP had new blood vessel density that was nearly two‐fold that of the CPC control (p < 0.05). In conclusion, CPC scaffold with autologous BMSC‐PRP doubled the new bone regeneration and blood vessel density in minipigs compared with the CPC control. In the present study, the new macroporous CPC system with co‐delivered BMSC‐PRP has been shown to promote scaffold resorption and bone regeneration in large defects. Copyright © 2017 John Wiley & Sons, Ltd.     

Form and functional repair of long bone using 3D‐printed bioactive scaffolds

Injuries to the extremities often require resection of necrotic hard tissue. For large‐bone defects, autogenous bone grafting is ideal but, similar to all grafting procedures, is subject to limitations. Synthetic biomaterial‐driven engineered healing offers an alternative approach. This work focuses on three‐dimensional (3D) printing technology of solid‐free form fabrication, more specifically robocasting/direct write. The research hypothesizes that a bioactive calcium‐phosphate scaffold may successfully regenerate extensive bony defects in vivo and that newly regenerated bone will demonstrate mechanical properties similar to native bone as healing time elapses. Robocasting technology was used in designing and printing customizable scaffolds, composed of 100% beta tri‐calcium phosphate (β‐TCP), which were used to repair critical sized long‐bone defects. Following full thickness segmental defects (~11 mm × full thickness) in the radial diaphysis in New Zealand white rabbits, a custom 3D‐printed, 100% β‐TCP, scaffold was implanted or left empty (negative control) and allowed to heal over 8, 12, and 24 weeks. Scaffolds and bone, en bloc, were subjected to micro‐CT and histological analysis for quantification of bone, scaffold and soft tissue expressed as a function of volume percentage. Additionally, biomechanical testing at two different regions, (a) bone in the scaffold and (b) in native radial bone (control), was conducted to assess the newly regenerated bone for reduced elastic modulus (E_r) and hardness (H) using nanoindentation. Histological analysis showed no signs of any adverse immune response while revealing progressive remodelling of bone within the scaffold along with gradual decrease in 3D‐scaffold volume over time. Micro‐CT images indicated directional bone ingrowth, with an increase in bone formation over time. Reduced elastic modulus (E_r) data for the newly regenerated bone presented statistically homogenous values analogous to native bone at the three time points, whereas hardness (H) values were equivalent to the native radial bone only at 24 weeks. The negative control samples showed limited healing at 8 weeks. Custom engineered β‐TCP scaffolds are biocompatible, resorbable, and can directionally regenerate and remodel bone in a segmental long‐bone defect in a rabbit model. Custom designs and fabrication of β‐TCP scaffolds for use in other bone defect models warrant further investigation.     

Phosphoserine‐modified calcium phosphate cements: bioresorption and substitution

This work reports the effects of phosphoserine addition on the biodegradability of calcium phosphate cements. The characteristics of a phosphoserine‐modified calcium phosphate cement without collagen in a large animal model are presented here for the first time. Critical size bone defects in the proximal tibia of 10 sheep were filled with the bone cement, and five sheep with empty defects were included as controls. The sheep were sacrificed after either 10 days or 12 weeks, and bones were processed for histological, histomorphometric and enzyme histochemical analyses as well as transmission electron microscopic examination. After 12 weeks, there was no significant reduction in either the implant or the bone defect cross‐sectional area. Different amounts of fibrous tissue were observed around the implant and in the bone defect after 12 weeks. The direct bone–implant contact decreased after 12 weeks (p = 0.034). Although the implanted material properly filled the defect and promoted an initial activation of macrophages and osteoblasts, the resorption and simultaneous substitution did not reach expected levels during the experimental time course. Although other studies have shown that the addition of phosphoserine to calcium phosphate cements that have already been modified with collagen I resulted in an acceleration of cement resorption and bone regeneration, this study demonstrates that phosphoserine‐modified calcium phosphate cements without collagen perform poorly in the treatment of bone defects. Efforts to use phosphoserine in the development of new composites should take into consideration the need to improve osteoconduction simultaneously via other means. Copyright © 2010 John Wiley & Sons, Ltd.     

Angiogenic and osteogenic regeneration in rats via calcium phosphate scaffold and endothelial cell co‐culture with human bone marrow mesenchymal stem cells (MSCs), human umbilical cord MSCs,human induced pluripotent stem cell‐derived MSCs and human embryonic stem cell‐derived MSCs

Angiogenesis is a limiting factor in regenerating large bone defects. The objective of this study was to investigate angiogenic and osteogenic effects of co‐culture on calcium phosphate cement (CPC) scaffold using human umbilical vein endothelial cells (hUVECs) and mesenchymal stem cells (MSCs) from different origins for the first time. hUVECs were co‐cultured with four types of cell: human umbilical cord MSCs (hUCMSCs), human bone marrow MSCs (hBMSCs) and MSCs from induced pluripotent stem cells (hiPSC‐MSCs) and embryonic stem cells (hESC‐MSCs). Constructs were implanted in 8 mm cranial defects of rats for 12 weeks. CPC without cells served as control 1. CPC with hBMSCs served as control 2. Microcapillary‐like structures were successfully formed on CPC in vitro in all four co‐cultured groups. Microcapillary lengths increased with time (p < 0.05). Osteogenic and angiogenic gene expressions were highly elevated and mineralization by co‐cultured cells increased with time (p < 0.05). New bone amount and blood vessel density of co‐cultured groups were much greater than controls (p < 0.05) in an animal study. hUVECs co‐cultured with hUCMSCs, hiPSC‐MSCs and hESC‐MSCs achieved new bone and vessel density similar to hUVECs co‐cultured with hBMSCs (p > 0.1). Therefore, hUCMSCs, hiPSC‐MSCs and hESC‐MSCs could serve as alternative cell sources to hBMSCs, which require an invasive procedure to harvest. In conclusion, this study showed for the first time that co‐cultures of hUVECs with hUCMSCs, hiPSC‐MSCs, hESC‐MSCs and hBMSCs delivered via CPC scaffold achieved excellent osteogenic and angiogenic capabilities in vivo. The novel co‐culture constructs are promising for bone reconstruction with improved angiogenesis for craniofacial/orthopaedic applications. Copyright © 2017 John Wiley & Sons, Ltd.     

Gelatin coating increases in vivo bone formation capacity of three‐dimensional 45S5 bioactive glass‐based crystalline scaffolds

Recent studies have demonstrated that surface characteristics, porosity, and mechanical strength of three‐dimensional 45S5‐type bioactive glass (BG)‐based scaffolds are directly correlated with osteogenic properties. Three‐dimensional BG‐based scaffolds obtained from maritime natural sponges (MNSs) as sacrificial templates exhibit the required morphological properties; however, no in vivo data about the osteogenic features are available. In this study, uncoated (Group A) and gelatin‐coated (Group B) crystalline MNS‐obtained BG‐based scaffolds were evaluated mechanically and seeded with human mesenchymal stem cells prior to subcutaneous implantation in immunodeficient mice. Before implantation and after explantation, micro‐computed tomography scans were conducted, and scaffolds were finally subjected to histomorphometry. Scaffolds of both groups showed bone formation. However, Group B scaffolds performed distinctly better as indicated by a significant increase in scaffold volume (8.95%, p = 0.039) over the implantation period compared with a nonsignificant increase of 5.26% in Group A scaffolds in micro‐computed tomography analysis. Furthermore, percentage bone area was 10.33% (±1.18%) in the Group B scaffolds, which was significantly (p = 0.007) higher compared with the 8.53% (±0.77%) in the Group A scaffolds in histomorphometry. Compressive strength was enhanced significantly by gelatin coating (9 ± 2 vs. 4 ± 1 MPa; p = 0.029). The presence of gelatin on the remnant parts was verified by scanning electron microscopy and X‐ray spectroscopy, demonstrating the coatings' resilience. MNS‐obtained BG‐based scaffolds were thus confirmed to exhibit osteogenic properties in vivo that can significantly be enhanced by gelatin coating.