Limited Bone Formation Research Articles

Bone augmentation using bioresorbable mesh domes containing bone graft granules

Bone graft granules are valuable tools for ridge area bone grafting owing to their ease of manipulation and interconnected porous structure. Guided bone regeneration (GBR) using barrier membranes is commonly used for alveolar ridge augmentation; however, the surgical procedures are technically complicated. In this study, we fabricated bioresorbable mesh domes (BMDs) using two types of Vicryl mesh (woven and knitted types) containing carbonate apatite granules. BMD samples were prepared in three groups: upper sides made from the woven type (UW) and lower sides made from the woven type (LW) (the UW/LW group), upper sides made from the woven type (UW) and lower sides made from the knitted type (LK) (the UW/LK group), and upper sides made from the knitted type (UK) and lower sides made from the knitted type (LK) (the UK/LK group). The samples were subsequently implanted into rabbit calvaria, and radiomorphometric and histological analyses were conducted. The UK/LK group exhibited enhanced appositional bone formation because the knitted mesh on the skin side prevented the infiltration of a substantial amount of fibrous tissue. This increase in bone formation could be attributed to the interaction between granules and osteoprogenitors that pass through the mesh from the host bone. Conversely, the UW/LW and UW/LK groups presented limited appositional bone formation. Compared with knitted mesh, woven mesh might tend to be absorbed over a short span, allowing fibrous tissue invasion and inhibiting new bone formation. Additionally, BMDs could retain granules in a targeted location and avoid displacement of the granules to unintended locations.Graphical

EXPLORING THE EXTRACELLULAR MATRIX FROM YOUNG DONORS TO ENHANCE THE IMPAIRED OSTEOGENIC PROPERTIES OF AGED CELLS

Mesenchymal stromal cells (MSC) have been proposed as an emerging cell therapy for bone tissue engineering applications. However, the healing capacity of the bone tissue is often compromised by patient's age and comorbidities, such as osteoporosis. In this context, it is important to understand the impact of donor age on the therapeutic potential of MSC. Importantly, the impact on donor age is not restricted to cells themselves but also to their microenvironment that is known to affect cell function.The extracellular matrix (ECM) has an important role in stem cell microenvironment, being able to modulate cell proliferation, self-renewal and differentiation. Decellularized cell-derived ECM (dECM) has been explored for regenerative medicine applications due to its bioactivity and its resemblance to the in vivo microenvironment. Thus, dECM offers the opportunity not only to develop microenvironments with customizable properties for improvement of cellular functions but also as a platform to study cellular niches in health and disease. In this study, we investigated the capacity of the microenvironment to rescue the impaired proliferative and osteogenic potential of aged MSC. The goal of this work was to understand if the osteogenic capacity of MSC could be modulated by exposure to a dECM derived from cells obtained from young donors. When aged MSC were cultured on dECM derived from young MSC, their in vitro proliferative and osteogenic capacities were enhanced. Our results suggest that the microenvironment, specifically the ECM, plays a crucial role in the osteogenic differentiation capacity of MSC. dECM might be a valuable clinical strategy to overcome the age-related decline in the osteogenic potential of MSC by recapitulating a younger microenvironment, attenuating the effects of aging on the stem cell niche. Overall, this study opens new possibilities for developing clinical strategies for elderly patients with limited bone formation capacity who currently lack effective treatments.Acknowledgements: The authors thank FCT for funding through the project DentalBioMatrix (PTDC/BTM-MAT/3538/2020) and to the research institutions iBB (UIDB/04565/2020 and UIDP/04565/2020) and Associate Laboratory i4HB (LA/P/0140/2020).

An osteoporosis bone defect regeneration strategy via three-dimension short fibers loaded with alendronate modified hydroxyapatite

Osteoporotic bone defect has become clinic challenge due to its morbid bone microenvironment. Overactive bone resorption and limited bone formation lead to unstable combination between bone tissue and scaffolds. Electrospinning has been widely used in guide tissue membrane, but its barrier property results in limited application. In order to optimize the structure and add anti-bone resorption function of electrospinning fibers, we exploited the application of short fibers generated by homogenization at osteoporotic tibial bone defect. The modified nano-hydroxyapatite (m-HA) was loaded with alendronate. It overcame the problem that hydrophilic drugs were difficult to distribute uniformly in hydrophobic fibers. We confirmed that m-HA was loaded into polycaprolactone (PCL) short fibers. PCL short fibers with m-HA (PCL/m-HA) continuously released ALN, provided stable structure and showed good cytocompatibility. In vitro, PCL/m-HA increased the activity of alkaline phosphatase (ALP), promoted extracellular matrix mineralization and upregulated the expression of osteogenesis-related genes, Col 1, Alp, osteopontin (Opn) and runt-related transcription factor 2 (Runx2). In vivo, PCL/m-HA short fibers accelerated the new bone formation, inhibited the bone resorption and rebalanced the bone microenvironment through regulating osteoprotegerin (OPG) /receptor activator of NF-kB (RANKL) ratio. The above results confirmed that the PCL/m-HA short fibers achieved the application of three-dimension osteoporotic bone defect and had potential prospects in bone tissue scaffolds.

In vivo evaluation of bone regeneration using ZIF8-modified polypropylene membrane in rat calvarium defects.

The profound potential of zeolitic imidazolate framework 8 (ZIF8) thin film for inducing osteogenesis has been previously established under in vitro conditions. As the next step towards the clinical application of ZIF8-modified substrates in periodontology, this in vivo study aimed to evaluate the ability of the ZIF8 crystalline layer to induce bone regeneration in an animal model defect. Following the mechanical characterization of the membranes and analysing the in vitro degradation of the ZIF8 layer, in vivo bone regeneration was evaluated in a critical-sized (5-mm) rat calvarial bone defect model. For each animal, one defect was randomly covered with either a polypropylene (PP) or a ZIF8-modified membrane (n = 7 per group), while the other defect was left untreated as a control. Eight weeks post surgery, bone formation was assessed by microcomputed tomography scanning, haematoxylin and eosin staining and immunohistochemical analysis. The ZIF8-modified membrane outperformed the PP membrane in terms of mechanical properties and revealed a trace Zn+2 release. Results of in vivo evaluation verified the superior barrier function of the ZIF8-coated membrane compared with pristine PP membrane. Compared with the limited marginal bone formation in the control and PP groups, the defect area was almost filled with mature bone in the ZIF8-coated membrane group. Our results support the effectiveness of the ZIF8-coated membrane as a promising material for improving clinical outcomes of guided bone regeneration procedures, without using biological components.

The Gradual Release of Alendronate for the Treatment of Critical Bone Defects in Osteoporotic and Control Rats.

Osteoporosis is a severe health problem with social and economic impacts on society. The standard treatment consists of the systemic administration of drugs such as bisphosphonates, with alendronate (ALN) being one of the most common. Nevertheless, complications of systemic administration occur with this drug. Therefore, it is necessary to develop new strategies, such as local administration. In this study, emulsion/dispersion scaffolds based on W/O emulsion of PCL and PF68 with ALN, containing hydroxyapatite (HA) nanoparticles as the dispersion phase were prepared using electrospinning. Scaffolds with different release kinetics were tested in vitro on the co-cultures of osteoblasts and osteoclast-like cells, isolated from adult osteoporotic and control rats. Cell viability, proliferation, ALP, TRAP and CA II activity were examined. A scaffold with a gradual release of ALN was tested in vivo in the bone defects of osteoporotic and control rats. The release kinetics were dependent on the scaffold composition and the used system of the poloxamers. The ALN was released from the scaffolds for more than 22 days. The behavior of cells cultured in vitro on scaffolds with different release kinetics was comparable. The difference was evident between cell co-cultures isolated from osteoporotic and control animals. The PCL/HA scaffold show slow degradation in vivo and residual scaffold limited new bone formation inside the defects. Nevertheless, the released ALN supported bone formation in the areas surrounding the residual scaffold. Interestingly, a positive effect of systemic administration of ALN was not proved. The prepared scaffolds enabled tunable control release of ALN. The effect of ALN was proved in vitro and in in vivo study supported peri-implant bone formation.

Histological Processing of CAD/CAM Titanium Scaffold after Long-Term Failure in Cranioplasty.

Cranioplasty is a frequently performed procedure after craniectomy and includes several techniques with different materials. Due to high overall complication rates, alloplastic implants are removed in many cases. Lack of implant material osseointegration is often assumed as a reason for failure, but no study has proven this in cranioplasty. This study histologically evaluates the osteointegration of a computer-aided design and computer-aided manufacturing (CAD/CAM) titanium scaffold with an open mesh structure used for cranioplasty. A CAD/CAM titanium scaffold was removed due to late soft tissue complications 7.6 years after cranioplasty. The histological analyses involved the preparation of non-decalcified slices from the scaffold’s inner and outer sides as well as a light-microscopic evaluation, including the quantification of the bone that had formed over the years. Within the scaffold pores, vital connective tissue with both blood vessels and nerves was found. Exclusive bone formation only occurred at the edges of the implant, covering 0.21% of the skin-facing outer surface area. The inner scaffold surface, facing towards the brain, did not show any mineralization at all. Although conventional alloplastic materials for cranioplasty reduce surgery time and provide good esthetic results while mechanically protecting the underlying structures, a lack of adequate stimuli could explain the limited bone formation found. CAD/CAM porous titanium scaffolds alone insufficiently osseointegrate in such large bone defects of the skull. Future research should investigate alternative routes that enable long-term osteointegration in order to reduce complication rates after cranioplasty. Opportunities could be found in mechano-biologically optimized scaffolds, material modifications, surface coatings, or other routes to sustain bone formation.

A Naringin-loaded gelatin-microsphere/nano-hydroxyapatite/silk fibroin composite scaffold promoted healing of critical-size vertebral defects in ovariectomised rat

In this study, we investigated the effect of three-dimensional of naringin/gelatin microspheres/nano-hydroxyapatite/silk fibroin (NG/GMs/nHA/SF) scaffolds on repair of a critical-size bone defect of lumbar 6 in osteoporotic rats. In this work, a cell-free scaffold for bone-tissue engineering based on a silk fibroin (SF)/nano-hydroxyapatite (nHA) scaffold was developed. The scaffold was fabricated by lyophilization. Naringin (NG) was loaded into gelatin microspheres (GMs), which were encapsulated in the nHA/SF scaffolds. The materials were characterized using x ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy and thermogravimetric analysis. Moreover, the biomechanics, degradation, and drug-release profile of the scaffold were also evaluated. In vitro, the effect of the scaffold on the adhesion, proliferation, and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) was evaluated. In vivo, at 3 months after ovariectomy, a critical-size lumbar defect was indued in the rats to evaluate scaffold therapeutic potential. A 3-mm defect in L6 developed in 60 SD rats, which were randomly divided into SF scaffold, nHA/SF scaffold, NG/nHA/SF scaffold, NG/GMs/nHA/SF scaffold, and blank groups (n = 12 each). At 4, 8, 12, and 16 weeks postoperatively, osteogenesis was evaluated by X-ray, micro-computed tomography, hematoxylin-eosin staining, and fast green staining, and by analysis of BMP-2, Runx2, and Ocn protein levels at 16 weeks. In our results, NG/GM/nHA/SF scaffolds exhibited good biocompatibility, biomechanical strength, and promoted BMSC adhesion, proliferation, and calcium nodule formation in vitro. Moreover, NG/GMs/nHA/SF scaffolds showed greater osteogenic differentiation potential than the other scaffolds in vitro. In vivo, gradual new bone formation was observed, and bone defects recovered by 16 weeks in the experimental group. In the blank group, limited bone formation was observed, and the bone defect was obvious. In conclusion, NG/GMs/nHA/SF scaffolds promoted repair of a lumbar 6 defect in osteoporotic rats. Therefore, the NG/GMs/nHA/SF biocomposite scaffold has potential as a bone-defect-filling biomaterial for bone regeneration.

Impact of Donor Age on the Osteogenic Supportive Capacity of Mesenchymal Stromal Cell-Derived Extracellular Matrix.

Mesenchymal stromal cells (MSC) have been proposed as an emerging cell-based therapeutic option for regenerative medicine applications as these cells can promote tissue and organ repair. In particular, MSC have been applied for the treatment of bone fractures. However, the healing capacity of these fractures is often compromised by patient’s age. Therefore, considering the use of autologous MSC, we evaluated the impact of donor age on the osteogenic potential of bone marrow (BM)-derived MSC. MSC from older patients (60 and 80 years old) demonstrated impaired proliferative and osteogenic capacities compared to MSC isolated from younger patients (30 and 45 years old), suggesting that aging potentially changes the quantity and quality of MSC. Moreover, in this study, we investigated the capacity of the microenvironment [i.e., extracellular matrix (ECM)] to rescue the impaired proliferative and osteogenic potential of aged MSC. In this context, we aimed to understand if BM MSC features could be modulated by exposure to an ECM derived from cells obtained from young or old donors. When aged MSC were cultured on decellularized ECM derived from young MSC, their in vitro proliferative and osteogenic capacities were enhanced, which did not happen when cultured on old ECM. Our results suggest that the microenvironment, specifically the ECM, plays a crucial role in the quality (assessed in terms of osteogenic differentiation capacity) and quantity of MSC. Specifically, the aging of ECM is determinant of osteogenic differentiation of MSC. In fact, old MSC maintained on a young ECM produced higher amounts of extracellularly deposited calcium (9.10 ± 0.22 vs. 4.69 ± 1.41 μg.μl–1.10–7 cells for young ECM and old ECM, respectively) and up-regulated the expression of osteogenic gene markers such as Runx2 and OPN. Cell rejuvenation by exposure to a functional ECM might be a valuable clinical strategy to overcome the age-related decline in the osteogenic potential of MSC by recapitulating a younger microenvironment, attenuating the effects of aging on the stem cell niche. Overall, this study provides new insights on the osteogenic potential of MSC during aging and opens new possibilities for developing clinical strategies for elderly patients with limited bone formation capacity who currently lack effective treatments.

CNT and rGO reinforced PMMA based bone cement for fixation of load bearing implants: Mechanical property and biological response

Polymethyl methacrylate (PMMA) bone cements (BCs) have some drawbacks, including limited bioactivity and bone formation, as well as inferior mechanical properties, which may result in failure of the BC. To deal with the mentioned issues, novel bioactive polymethyl methacrylate-hardystonite (PMMA-HT) bone cement (BC) reinforced with 0.25 and 0.5 wt% of carbon nanotube (CNT) and reduced graphene oxide (rGO) was synthesized. In this context, the obtained bone cements were evaluated in terms of their mechanical and biological characteristics. The rGO reinforced bone cement exhibited better mechanical properties to the extent that the addition of 0.5 wt% of rGO where its compressive and tensile strength of bioactive PMMA-HT/rGO cement escalated from 92.07 ± 0.72 MPa, and 40.02 ± 0.71 MPa to 187.48 ± 5.79 MPa and 64.92 ± 0.75 MPa, respectively. Besides, the mechanisms of toughening, apatite formation, and cell interaction in CNT and rGO encapsulated PMMA have been studied. Results showed that the existence of CNT and rGO in BCs led to increase of MG63 osteoblast viability, and proliferation. However, rGO reinforced bone cement was more successful in supporting MG63 cell attachment compared to the CNT counterpart due to its wrinkled surface, which made a suitable substrate for cell adhesion. Based on the results, PMMA-HT/rGO can be a proper bone cement for the fixation of load-bearing implants.

Endochondral Bone Regeneration by Non-autologous Mesenchymal Stem Cells.

Mimicking endochondral bone formation is a promising strategy for bone regeneration. To become a successful therapy, the cell source is a crucial translational aspect. Typically, autologous cells are used. The use of non-autologous mesenchymal stromal cells (MSCs) represents an interesting alternative. Nevertheless, non-autologous, differentiated MSCs may trigger an undesired immune response, hampering bone regeneration. The aim of this study was to unravel the influence of the immune response on endochondral bone regeneration, when using xenogeneic (human) or allogeneic (Dark Agouti) MSCs. To this end, chondrogenically differentiated MSCs embedded in a collagen carrier were implanted in critical size femoral defects of immunocompetent Brown Norway rats. Control groups were included with syngeneic/autologous (Brown Norway) MSCs or a cell-free carrier. The amount of neo-bone formation was proportional to the degree of host-donor relatedness, as no full bridging of the defect was observed in the xenogeneic group whereas 2/8 and 7/7 bridges occurred in the allogeneic and the syngeneic group, respectively. One week post-implantation, the xenogeneic grafts were invaded by pro-inflammatory macrophages, T lymphocytes, which persisted after 12 weeks, and anti-human antibodies were developed. The immune response toward the allogeneic graft was comparable to the one evoked by the syngeneic implants, aside from an increased production of alloantibodies, which might be responsible for the more heterogeneous bone formation. Our results demonstrate for the first time the feasibility of using non-autologous MSC-derived chondrocytes to elicit endochondral bone regeneration in vivo. Nevertheless, the pronounced immune response and the limited bone formation observed in the xenogeneic group undermine the clinical relevance of this group. On the contrary, although further research on how to achieve robust bone formation with allogeneic cells is needed, they may represent an alternative to autologous transplantation.

Collagen type X is essential for successful mesenchymal stem cell-mediated cartilage formation and subsequent endochondral ossification.

in tissue engineering, endochondral ossification (EO) is often replicated by chondrogenically differentiating mesenchymal stromal cells (MSCs) in vitro and achieving bone formation through in vivo implantation. The resulting marrow-containing bone constructs are promising as a treatment for bone defects. However, limited bone formation capacity has prevented them from reaching their full potential. This is further complicated since it is not fully understood how this bone formation is achieved. Acellular grafts derived from chondrogenically differentiated MSCs can initiate bone formation; however, which component within these decellularised matrices contribute to bone formation has yet to be determined. Collagen type X (COLX), a hypertrophy-associated collagen found within these constructs, is involved in matrix organisation, calcium binding and matrix vesicle compartmentalisation. However, the importance of COLX during tissue-engineered chondrogenesis and subsequent bone formation is unknown. The present study investigated the importance of COLX by shRNA-mediated gene silencing in primary MSCs. A significant knock-down of COLX disrupted the production of extracellular matrix key components and the secretion profile of chondrogenically differentiated MSCs. Following in vivo implantation, disrupted bone formation in knock-down constructs was observed. The importance of COLX was confirmed during both chondrogenic differentiation and subsequent EO in this tissue engineered setting.

Exosomes Secreted by Stem Cells from Human Exfoliated Deciduous Teeth Promote Alveolar Bone Defect Repair through the Regulation of Angiogenesis and Osteogenesis

Exosomes are important mediators of intercellular communication and have a vital part in the diagnosis and treatment of various diseases in humans. Here, we investigated the benefits and underlying mechanism of exosomes secreted via stem cells from human exfoliated deciduous teeth (SHED-derived exosomes) in promoting alveolar bone regeneration, thus providing new insights into exosome-based therapy for periodontitis. SHED-derived exosomes were isolated by ultracentrifugation. The impacts of SHED-derived exosomes on the angiogenic ability of human umbilical vein endothelial cells (HUVECs) and the osteogenic capability of rat bone marrow mesenchymal stem cells (BMSCs) were evaluated in vitro. Compound C, a pharmacological blocker of adenosine monophosphate-activated protein kinase (AMPK), was used to examine the role of the AMPK signaling cascade in these processes. Periodontal defect rat models were established and treated with PBS, β-tricalcium phosphate (β-TCP), or a grouping of exosomes/β-TCP. Microcomputed tomography (micro-CT) scanning, hematoxylin and eosin (HE) staining, Masson staining, and immunofluorescence staining were done to inspect the impacts of the exosomes/β-TCP combination on periodontal bone regeneration. Our outcomes indicated that the expression of angiogenesis-related genes (KDR, SDF-1, and FGF2), osteogenesis-related genes (COL1, RUNX2, and OPN), and phosphorylated (p)-AMPK were upregulated after treatment with exosomes, while the positive impacts of SHED-derived exosomes on HUVECs and BMSCs were partially reversed by compound C. Micro-CT analysis demonstrated that the exosomes/β-TCP group exhibited better bone regeneration than either the β-TCP group or the control group. Additionally, the results of HE and Masson staining as well as immunofluorescence staining showed neovascularization and new bone formation in the exosomes/β-TCP group but only limited new bone formation in the other two groups. Thus, SHED-derived exosomes contribute to periodontal bone regeneration by promoting neovascularization and new bone formation, possibly through the AMPK signaling pathway.

Effect of pore size in bone regeneration using polydopamine-laced hydroxyapatite collagen calcium silicate scaffolds fabricated by 3D mould printing technology.

The pore size of the scaffold is a critical factor in repairing large bone defect. Here, we investigated the potential of bone regeneration using novel nanocomposite polydopamine-laced hydroxyapatite collagen calcium silicate (HCCS-PDA) scaffolds with two different pore sizes, 250 and 500μm. A total of 12 male Sprague-Dawley rats were implanted with HCCS-PDA scaffold with pore size of either 250 or 500μm into surgically created critical-sized defect (CSD). HCCS-PDA scaffolds were fabricated using mould printing technique. The effect of pore size on mechanical strength of the scaffolds was assessed by compression testing. After seeding with rat mesenchymal stem cells (rMSCs), the scaffolds were implanted, and new bone formation was evaluated using microCT and histomorphometric analysis after 8weeks. MicroCT and histology analysis demonstrated restricted peripheral new bone formation in either dural or periosteal side and limited new bone formation in the 250μm pore scaffold. Conversely, the 500-μm pore scaffold showed more penetration of new bone into the scaffold and greater bone regeneration in the rat CSD. Based on our results, which demonstrated improved new bone formation in 500μm pores scaffold, we can conclude that effective scaffold pore size that induces osteointegration and bone regeneration is around 500μm for HCCS-PDA nanocomposite scaffold.

Physical and Histological Comparison of Hydroxyapatite, Carbonate Apatite, and β-Tricalcium Phosphate Bone Substitutes.

Three commercially available artificial bone substitutes with different compositions, hydroxyapatite (HAp; Neobone®), carbonate apatite (CO3Ap; Cytrans®), and β-tricalcium phosphate (β-TCP; Cerasorb®), were compared with respect to their physical properties and tissue response to bone, using hybrid dogs. Both Neobone® (HAp) and Cerasorb® (β-TCP) were porous, whereas Cytrans® (CO3Ap) was dense. Crystallite size and specific surface area (SSA) of Neobone® (HAp), Cytrans® (CO3Ap), and Cerasorb® (β-TCP) were 75.4 ± 0.9 nm, 30.8 ± 0.8 nm, and 78.5 ± 7.5 nm, and 0.06 m2/g, 18.2 m2/g, and 1.0 m2/g, respectively. These values are consistent with the fact that both Neobone® (HAp) and Cerasorb® (β-TCP) are sintered ceramics, whereas Cytrans® (CO3Ap) is fabricated in aqueous solution. Dissolution in pH 5.3 solution mimicking Howship’s lacunae was fastest in CO3Ap (Cytrans®), whereas dissolution in pH 7.3 physiological solution was fastest in β-TCP (Cerasorb®). These results indicated that CO3Ap is stable under physiological conditions and is resorbed at Howship’s lacunae. Histological evaluation using hybrid dog mandible bone defect model revealed that new bone was formed from existing bone to the center of the bone defect when reconstructed with CO3Ap (Cytrans®) at week 4. The amount of bone increased at week 12, and resorption of the CO3Ap (Cytrans®) was confirmed. β-TCP (Cerasorb®) showed limited bone formation at week 4. However, a larger amount of bone was observed at week 12. Among these three bone substitutes, CO3Ap (Cytrans®) demonstrated the highest level of new bone formation. These results indicate the possibility that bone substitutes with compositions similar to that of bone may have properties similar to those of bone.

Chemical Self-Assembly of Multifunctional Hydroxyapatite with a Coral-like Nanostructure for Osteoporotic Bone Reconstruction.

Bone defects/fractures are common in older people suffering from osteoporosis. Traditional hydroxyapatite (HA) materials for osteoporotic bone repair face many challenges, including limited bone formation and aseptic loosening of orthopedic implants. In this study, a new multifunctional HA is synthesized by spontaneous assembly of alendronate (AL) and Fe3O4 onto HA nanocrystals for osteoporotic bone regeneration. The chemical coordination of AL and Fe3O4 with HA does not induce lattice deformation, resulting in a functionalized HA (Func-HA) with proper magnetic property and controlled release manner. The Func-HA nanocrystals have been encapsulated in polymer substrates to further investigate their osteogenic capability. In vitro and in vivo evaluations reveal that both AL and Fe3O4, especially the combination of two functional groups on HA, can inhibit osteoclastic activity and promote osteoblast proliferation and differentiation, as well as enhance implant osseointegration and accelerate bone remodeling under osteoporotic condition. The as-developed Func-HA with coordinating antiresorptive ability, magnetic property, and osteoconductivity might be a desirable biomaterial for osteoporotic bone defect/fracture treatment.

Novel alginate biphasic scaffold for osteochondral regeneration: an in vivo evaluation in rabbit and sheep models.

Current therapeutic strategies for osteochondral restoration showed a limited regenerative potential. In fact, to promote the growth of articular cartilage and subchondral bone is a real challenge, due to the different functional and anatomical properties. To this purpose, alginate is a promising biomaterial for a scaffold-based approach, claiming optimal biocompatibility and good chondrogenic potential. A previously developed mineralized alginate scaffold was investigated in terms of the ability to support osteochondral regeneration both in a large and medium size animal model. The results were evaluated macroscopically and by microtomography, histology, histomorphometry, and immunohistochemical analysis. No evidence of adverse or inflammatory reactions was observed in both models, but limited subchondral bone formation was present, together with a slow scaffold resorption time.The implantation of this biphasic alginate scaffold provided partial osteochondral regeneration in the animal model. Further studies are needed to evaluate possible improvement in terms of osteochondral tissue regeneration for this biomaterial.

Functionalization of PCL-3D electrospun nanofibrous scaffolds for improved BMP2-induced bone formation

Bone morphogenic protein 2 (BMP2) is a key growth factor for bone regeneration, possessing FDA approval for orthopedic applications. BMP2 is often required in supratherapeutic doses clinically, yielding adverse side effects and substantial treatment costs. Considering the crucial role of materials for BMPs delivery and cell osteogenic differentiation, we devote to engineering an innovative bone-matrix mimicking niche to improve low dose of BMP2-induced bone formation. Our previous work describes a novel technique, named thermally induced nanofiber self-agglomeration (TISA), for generating 3D electrospun nanofibrous (NF) polycaprolactone (PCL) scaffolds. TISA process could readily blend PCL with PLA, leading to increased osteogenic capabilities in vitro, however, these bio-inert synthetic polymers produced limited BMP2-induced bone formation in vivo. We therefore hypothesize that functionalization of NF 3D PCL scaffolds with bone-like hydroxyapatite (HA) and BMP2 signaling activator phenamil will provide a favorable osteogenic niche for bone formation at low doses of BMP2. Compared to PCL-3D scaffolds, PCL/HA-3D scaffolds demonstrated synergistically enhanced osteogenic differentiation capabilities of C2C12 cells with phenamil. Importantly, in vivo studies showed that this synergism was able to generate significantly increased new bone in an ectopic mouse model, suggesting that PCL/HA-3D scaffolds act as a favorable synthetic extracellular matrix for bone regeneration.

Bone Loss in Rheumatoid Arthritis: Basic Mechanisms and Clinical Implications.

Patients with rheumatoid arthritis (RA) have historically developed progressive damage of articular bone and cartilage, which correlates with disability over time. In addition, these patients are prone to periarticular and systemic bone loss, carrying additional morbidity. In contrast to what is seen in many other rheumatic diseases, the impact of inflammation on bone in RA is uniquely destructive. Loss of articular bone (erosions) and periarticular bone (demineralization) is a result of excessive bone resorption and markedly limited bone formation. There has been tremendous progress in preventing net bone loss in RA with the advent of disease-modifying agents, including biologic agents and small molecules, that both limit inflammation and may have a direct impact on the prevention of cytokine- and antibody-driven osteoclastogenesis. However, repair of existing bone erosions, although feasible, is observed infrequently. Lack of repair is a consequence of suppression of osteoblast function and bone formation by some of the same mechanisms that promote osteoclastogenesis and bone resorption. As new agents are introduced to control inflammation in RA, and novel mechanisms to target synovitis are identified, it may be possible in the future to fully repair damaged bone.

Composite scaffolds loaded with bone mesenchymal stem cells promote the repair of radial bone defects in rabbit model

This study aimed to investigate the efficacy of three-dimensional scaffolds of silk fibroin/chitosan/nano-hydroxyapatite (SF/CS/nHA) and bone marrow derived mesenchymal stem cells (BMSCs) on the repair of long segmental bone defects in rabbits. BMSCs were cultured with SF/CS/nHA in vitro, and cell proliferation, alkaline phosphatase activity and Ca2+ content were examined. A 15mm segmental defect in the radius was generated in 12 New Zealand White rabbits, which were divided randomly into three groups (n=4): experimental group with SF/CS/nHA scaffold of induced BMSCs; control group with SF/CS/nHA scaffold; and blank group without any materials. Postoperatively at 12 weeks, osteogenesis effect and the degradation and absorption of SF/CS/nHA were evaluated by X-ray, hematoxylin eosin staining, and scanning electron microscopy. In vitro, SF/CS/nHA scaffolds exhibited good biocompatibility and no toxicity. SF/CS/nHA promoted adhesion, growth, and calcium nodule formation of BMSCs compared to control (P<0.05). In vivo, we observed gradual new bone formation and bone defect gradually recovered at 12 weeks in experimental and control group, but more new bone was formed in experimental group (P<0.05). In blank group, limited bone formation was observed and bone defect was obvious. In conclusion, SF/CS/nHA scaffolds loaded with BMSCs achieve high efficacy to repair segmental defect in the radius.

Biomimetic Functionalized Surfaces and the Induction of Bone Formation.

Tissue engineering still needs to assign the molecular basis of pattern formation, tissue induction, and morphogenesis: What next to morphogens and stem cells? Macroporous biomimetic matrices per se, without the addition of the soluble osteogenic molecular signals of the transforming growth factor-β (TGF-β) supergene family, remarkably initiate the induction of bone formation. Carving geometries within different calcium phosphate-based macroporous bioreactors we show that geometric cues imprinted within the macroporous spaces initiate the spontaneous induction of bone. Concavities biomimetize the remodeling cycle of the primate osteonic bone and are endowed with functionalized smart geometric cues that per se initiate osteoblasts' differentiation with the expression and secretion of osteogenic molecular signals that induce bone as a secondary response. To study the role of calcium ions (Ca++) and osteoclastogenesis, coral-derived calcium carbonate (CC)/hydroxyapatite (HA) bioreactors with limited conversion to HA (7% HA/CC) were preloaded with 500 μg of the L-type voltage gated calcium channel blocker verapamil hydrochloride. Bioreactors were also loaded with 240 μg of the bisphosphonate zoledronate, an osteoclast inhibitor, and implanted in heterotopic sites of the rectus abdominis muscle of Papio ursinus. Bisphosphonate-treated specimens were characterized by a delayed profoundly inhibited induction of tissue patterning with limited induction of bone. Macroporous constructs pretreated with verapamil hydrochloride yielded limited bone formation. Similarly, 125 or 150 μg human Noggin previously adsorbed onto the macroporous bioreactors resulted in minimal bone formation by induction, indirectly showing that the initiation of bone formation is through the bone morphogenetic protein (BMP) pathway. Downregulation of BMP-2 and osteogenic protein-1 (OP-1) with upregulation of Noggin correlated with limited bone induction. Angiogenesis, capillary sprouting, and Ca++ provide chemotactic signals for myoendothelial, myoblastic, and pericytic stem cell differentiation into osteoblastic-like cells expressing the osteogenic soluble molecular signals of the TGF-β supergene family. Secreted gene products are embedded directly onto the substratum within its regulatory concavities. The protected microenvironment of the concavities biomimetizes the phylogenetically ancient repetitive multitested designs and topographies of Nature. Migrating cells onto the primed substratum by osteoclastic nanotopographical geometric inductive modifications convert geometrical cues set by osteoclastogenesis into BMP gene expression pathways that ultimately set into motion the spontaneous induction of bone formation.