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Recent advances have revived interest in the concept of osteocyte perilacunar/canalicular remodeling (PLR) and have motivated efforts to identify the mechanisms regulating this process in bone in the context of normal physiology and pathological conditions. Here, we describe several methods that are evaluating morphological changes associated with PLR function of osteocytes.

We assessed the combined impacts of human demineralized bone matrix (hDBM) scaffold, adipose-derived stem cells (hADS), and photobiomodulation (PBM) on bone repair of a critical size femoral defect (CSFD) in 72 rats. The rats were divided into six groups: control (group 1); ADS (group 2 - ADS transplanted into hDBM); PBM (group 3 - PBM-treated CSFDs); ADS + PBM in vivo (group 4 - ADS transplanted into hDBM and the CSFDs were treated with PBM in vivo); ADS + PBM in vitro (group 5 - ADS were treated with PBM in vitro, then seeded into hDBM); and ADS + PBM in vitro+in vivo (group 6 - PBM-treated ADS were seeded into hDBM, and the CSFDs were treated with PBM in vivo. At the anabolic phase (2 weeks after surgery), bone strength parameters of the groups 5, 6, and 4 were statistically greater than the control, ADS, and PBM in vivo groups (all, p = 0.000). Computed tomography (CT) scans during the catabolic phase (6 weeks after surgery) of bone healing revealed that the Hounsfield unit (HU) of CSFD in the groups 2 (p = 0.000) and 5 (p = 0.019) groups were statistically greater than the control group. The groups 5, 4, and 6 had significantly increased bone strength parameters compared with the PBM in vivo, control, and ADS groups (all, p = 0.000). The group 5 was statistically better than the groups 4, and 6 (both, p = 0.000). In vitro preconditioned of hADS with PBM significantly increased bone repair in a rat model of CSFD in vivo., 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 © 2020 Elsevier Inc. All rights reserved.)

Extracellular matrices (ECMs) derived from native tissues/organs have been used as biomaterials for tissue engineering and regenerative medicine in a wide range of preclinical and clinical settings. The success or failure of these applications is largely contingent on the host responses to the matrices in vivo. Despite retaining their native structural and functional proteins, bone ECM-based transplants have been reported to evoke adverse immune responses in many cases; thus, optimizing the immunomodulatory properties of bone ECMs is critical for ensuring downstream regenerative outcomes. Using a simple digestion-neutralization protocol, we transformed the commonly used bone-derived filler particles into gel bioscaffolds. Instead of inducing macrophages toward proinflammatory (M1) polarization, as reported in the literature and confirmed in the present study for ECM particles, the ECM gels were found to be more likely to polarize macrophages toward regulatory/anti-inflammatory (M2) phenotypes, leading to enhanced tissue regeneration in a rat periodontal defect model. The present work demonstrates a simple, practical and economical strategy to modify the immunomodulatory properties of bone ECMs before their in vivo transplantation and hence has important implications that may facilitate the use of ECM-based bioscaffolds derived from diverse sources of tissues for regenerative purposes., (Copyright © 2019. Published by Elsevier B.V.)

Purpose of Review: Osteocytes are the most abundant bone cells. They are completely encased in mineralized tissue, sitting inside lacunae that are connected by a multitude of canaliculi. In recent years, the osteocyte network has been shown to fulfill endocrine functions and to communicate with a number of other organs. This review addresses emerging knowledge on the connectome of the lacunocanalicular network in different types of bone tissue., Recent Findings: Recent advances in three-dimensional imaging technology started to reveal parameters that are well known from general theory to characterize the function of networks, such as network density, degree of nodes, or shortest path length through the network. The connectome of the lacunocanalicular network differs in some aspects between lamellar and woven bone and seems to change with age. More research is needed to relate network structure to function, such as intercellular transport or communication and its role in mechanosensation, as well as to understand the effect of diseases.

Purpose of Review: In perilacunar/canalicular remodeling (PLR), osteocytes dynamically resorb, and then replace, the organic and mineral components of the pericellular extracellular matrix. Given the enormous surface area of the osteocyte lacuna-canalicular network (LCN), PLR is important for maintaining homeostasis of the skeleton. The goal of this review is to examine the motivations and critical considerations for the analysis of PLR, in both in vitro and in vivo systems., Recent Findings: Morphological approaches alone are insufficient to elucidate the complex mechanisms regulating PLR in the healthy skeleton and in disease. Understanding the role and regulation of PLR will require the incorporation of standardized PLR outcomes as a routine part of skeletal phenotyping, as well as the development of improved molecular and cellular outcomes. Current PLR outcomes assess PLR enzyme expression, the LCN, and bone matrix composition and organization, among others. Here, we discuss current PLR outcomes and how they have been applied to study PLR induction and suppression in vitro and in vivo. Given the role of PLR in skeletal health and disease, integrated analysis of PLR has potential to elucidate new mechanisms by which osteocytes participate in skeletal health and disease.

Due to the similar collagen composition and closely physiological relationship with soft connective tissues, demineralized bone matrices (DBMs) were used to repair the injured tendon or ligament. However, the osteoinductivity of DBMs would be a huge barrier of these applications. Hydrogen peroxide (H 2 O 2 ) has been proved to reduce the osteoinductivity of DBMs. Nevertheless, the biological properties of H 2 O 2 -treated DBMs have not been evaluated completely, while the potential mechanism of H 2 O 2 compromising osteoinductivity is also unclear. Hence, the purpose of this study was to characterize the biological properties of H 2 O 2 -treated DBMs and search for the proof that H 2 O 2 could compromise osteoinductivity of DBMs. Decellularized and demineralized bone matrices (DCDBMs) were washed by 3% H 2 O 2 for 12 h to fabricate the H 2 O 2 -treated DCDBMs (HPTBMs). Similar biological properties including collagen, biomechanics, and biocompatibility were observed between DCDBMs and HPTBMs. The immunohistochemistry staining of bone morphogenetic protein 2 (BMP-2) was negative in HPTBMs. Furthermore, HPTBMs exhibited significantly reduced osteoinductivity both in vitro and in vivo. Taken together, these findings suggest that the BMP-2 in DCDBMs could be the target of H 2 O 2 . HPTBMs could be expected to be used as a promising scaffold for tissue engineering. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019., (© 2019 Wiley Periodicals, Inc.)

With ageing and various diseases, the vascular pore volume fraction (porosity) in cortical bone increases, and the morphology of the pore network is altered. Cortical bone elasticity is known to decrease with increasing porosity, but the effect of the microstructure is largely unknown, while it has been thoroughly studied for trabecular bone. Also, popular micromechanical models have disregarded several micro-architectural features, idealizing pores as cylinders aligned with the axis of the diaphysis. The aim of this paper is to quantify the relative effects on cortical bone anisotropic elasticity of porosity and other descriptors of the pore network micro-architecture associated with pore number, size and shape. The five stiffness constants of bone assumed to be a transversely isotropic material were measured with resonant ultrasound spectroscopy in 55 specimens from the femoral diaphysis of 29 donors. The pore network, imaged with synchrotron radiation X-ray micro-computed tomography, was used to derive the pore descriptors and to build a homogenization model using the fast Fourier transform (FFT) method. The model was calibrated using experimental elasticity. A detailed analysis of the computed effective elasticity revealed in particular that porosity explains most of the variations of the five stiffness constants and that the effects of other micro-architectural features are small compared to usual experimental errors. We also have evidence that modelling the pore network as an ensemble of cylinders yields biased elasticity values compared to predictions based on the real micro-architecture. The FFT homogenization method is shown to be particularly efficient to model cortical bone.

Mandibular defects, caused by congenital, pathological or iatrogenic insults, can significantly affect patient quality of life. The reconstruction of mandible has recently gained the interest of clinical and tissue engineering researchers. The purpose of this study was to evaluate the effectiveness of three-dimensional (3-D) cultured autologous grafts prepared using bone marrow-derived mesenchymal stem cells (BMSCs) combined with demineralized bone matrix (DBM) scaffolds for the restoration of mandibular defects. Cylindrical defects were created in the mandibular body of minipigs and filled with 3D-cultured BMSCs/DBM autografts, 2D-cultured BMSCs/DBM autografts, DBM material (without cells), or were left unfilled (blank). Using computed tomographic (CT) imaging and histological staining, we found that treatment of mandibular defects using 3-D cultured BMSCs/DBM autografts offered improvements in bone formation over both 2-D cultured autografts and cell-free DBM scaffolds. We found increased osteoid formation in 3D and 2D cultures, with more osteogenic cells present in the 3D constructs. We suggest that 3-D cultured homograft BMSCs combined with DBM scaffolds represents a new strategy for bone reconstruction, with potential future clinical applicability.

Background: Gene-activated matrix (GAM) induces sustained local production of growth factors to promote tissue regeneration. GAM contains a plasmid DNA (pDNA) encoding target proteins that is physically entrapped within a biodegradable matrix carrier. GAM with a pDNA encoding the first 34 amino acids of parathyroid hormone (PTH 1-34) and a collagen matrix enhances bone regeneration in long bone defects. Demineralized freeze-dried bone allograft (DFDBA) is a widely used osteoinductive bone graft. The present study determined the osteogenic effects of PTH-GAM with a collagen or DFDBA/collagen composite (D/C) matrix for treating craniofacial bone defects., Methods: We constructed a pDNA encoding human PTH 1-34 and performed cyclic AMP ELISA to verify the bioactivity of PTH 1-34. Next, we generated a D/C matrix and PTH-GAMs containing a collagen matrix (PTH-C-GAM) or D/C matrix (PTH-D/C-GAM). Rats with critical-sized calvarial bone defects were divided into four groups, namely, untreated rats (sham group) and rats grafted with D/C matrix, PTH-C-GAM, or PTH-D/C-GAM (D/C, PTH-C-GAM, or PTH-D/C-GAM groups, respectively). PTH expression was determined by performing immunohistochemical staining after 4 and 8 weeks. New bone formation was evaluated by performing radiography, dual-energy X-ray absorptiometry, microcomputed tomography, and histological examination., Results: PTH pDNA-transfected cells secreted bioactive PTH 1-34. Moreover, PTH was expressed at 4 and 8 weeks after the surgery in rats in the PTH-C-GAM group but not in rats in the D/C group. New bone formation in the calvarial bone defects, from more to less, was in the order of PTH-D/C-GAM, PTH-C-GAM, D/C, and sham groups., Conclusion: Our results indicate that PTH-GAM with a collagen matrix promotes local PTH production for at least 8 weeks and bone regeneration in craniofacial bone defect. Moreover, our results indicate that replacement of the collagen matrix with the D/C matrix improves the osteogenic effects of PTH-GAM., (Copyright © 2018. Published by Elsevier Taiwan LLC.)

Demineralized bone matrix (DBM), as an extracellular matrix (ECM), has had limited use as a medical replacement although studies have reported a possibility for its use in tendon or ligament tissue engineering. To be an acid-extracted organic matrix, DBM contains much of bone protein, with a small amount of inorganic solids and some cell debris. However, cell debris is a critical factor that triggers inflammatory reaction in clinical reconstructions using ECM. In this study, we used a protocol incorporating the use of detergent with nuclease treatment to prepare decellularized DBM (DCDBM). DNA quantification analysis and histological observation confirmed that cells were completely removed from DBM. The inherent ultrastructure of DBM was well preserved after decellularization as observed through scanning electron microscopy. Additionally, calcium and phosphorus were absent and the specific functional groups of collagen remained after decellularization. Moreover, 79.71% of the tensile strength of DBM was retained and the viscoelastic properties were similar to the ligament. Furthermore, DCDBM promoted the adhesion and proliferation of NIH-3T3 fibroblasts in vitro and triggered less inflammation response at 12 weeks subcutaneous implantation in a rat model. These results demonstrate that the DCDBM has the potential to be used for tendon and ligament replacement. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 468-478, 2018., (© 2017 Wiley Periodicals, Inc.)

Decellularized bone matrix is gaining a lot of attention as implantable biomaterials and/or biological scaffolds for bone tissue repair, and shows good clinical performance. This chapter describes the processing techniques and characterization protocols of decellularized bone. For the applications of the decellularized bone scaffold in promoting bone repair and regeneration, we discuss some of the current advances, and highlight the advantages and disadvantages of these scaffolds. Fabrication and application of the hydrogel derived from decellularized bone for bone tissue engineering are also presented.

Osteocytes-the central regulators of bone remodeling-are enclosed in a network of microcavities (lacunae) and nanocanals (canaliculi) pervading the mineralized bone. In a hitherto obscure process related to aging and disease, local plugs in the lacuno-canalicular network disrupt cellular communication and impede bone homeostasis. By utilizing a suite of high-resolution imaging and physics-based techniques, it is shown here that the local plugs develop by accumulation and fusion of calcified nanospherites in lacunae and canaliculi (micropetrosis). Two distinctive nanospherites phenotypes are found to originate from different osteocytic elements. A substantial deviation in the spherites' composition in comparison to mineralized bone further suggests a mineralization process unlike regular bone mineralization. Clearly, mineralization of osteocyte lacunae qualifies as a strong marker for degrading bone material quality in skeletal aging. The understanding of micropetrosis may guide future therapeutics toward preserving osteocyte viability to maintain mechanical competence and fracture resistance of bone in elderly individuals., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The formation of the complex osteocytic network relies on the emission of long cellular processes involved in communication, mechanical strain sensing, and bone turnover control. Newly deposited osteocytic processes rapidly become trapped within the calcifying matrix, and, therefore, they must adopt their definitive conformation and contact their targets in a single morphogenetic event. However, the cellular mechanisms ensuring the robustness of this unique mode of morphogenesis remain unknown. To address this issue, we examined the developing calvaria of the amphibian Xenopus tropicalis by confocal, two-photon, and super-resolution imaging, and described flattened osteocytes lying within a woven bone structured in lamellae of randomly oriented collagen fibers. While most cells emit peripheral and perpendicular processes, we report two osteocytes morphotypes, located at different depth within the bone matrix and exhibiting distinct number and orientation of perpendicular cell processes. We show that this pattern is conserved with the chick Gallus gallus and suggest that the cellular microenvironment, and more particularly cell-cell contact, plays a fundamental role in the induction and stabilization of osteocytic processes. We propose that this intrinsic property might have been evolutionarily selected for its ability to robustly generate self-organizing osteocytic networks harbored by the wide variety of bone shapes and architectures found in extant and extinct vertebrates., (© 2016 Wiley Periodicals, Inc.)

Several decades ago, aluminum encephalopathy associated with osteomalacia has been recognized as the major complication of chronic renal failure in dialyzed patients. Removal of aluminum from the dialysate has led to a disappearance of the disease. However, aluminum deposit occurs in the hydroxyapatite of the bone matrix in some clinical circumstances that are presented in this review. We have encountered aluminum in bone in patients with an increased intestinal permeability (coeliac disease), or in the case of prolonged administration of aluminum anti-acid drugs. A colocalisation of aluminum with iron was also noted in cases of hemochromatosis and sickle cell anemia. Aluminium was also identified in a series of patients with exostosis, a frequent benign bone tumor. Corrosion of prosthetic implants composed of grade V titanium (TA6V is an alloy containing 6% aluminum and 4% vanadium) was also observed in a series of hip or knee revisions. Aluminum can be identified in undecalcified bone matrix stained by solochrome azurine, a highly specific stain allowing the detection of 0.03 atomic %. Colocalization of aluminum and iron does not seem to be the fruit of chance but the cellular and molecular mechanisms are still poorly understood. Histochemistry is superior to spectroscopic analyses (EDS and WDS in scanning electron microscopy)., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Purpose: To investigate the effects of locally applied simvastatin plus biphasic calcium phosphate (BoneCeramic(r)) or collagen sponge on bone formation in critical-sized bone defects., Methods: Thirty defects of 5mm in diameter were created bilaterally with a trephine bur in the calvariae of fifteen Wistar rats. The defects were divided into five groups: group 1 - control, no treatment; group 2 (BoneCeramic(r)); group 3 (BoneCeramic(r) + 0.1mg simvastatin); group 4 (collagen sponge); and group 5 (collagen sponge + 0.1mg simvastatin). After eight weeks the animals were euthanized and their calvariae were histologically processed. Hematoxylin and eosin-stained sections were subjected to histological and histomorphometrical analyses. The area of newly formed bone was calculated and compared between groups., Results: The greater amount of a bone-like tissue was formed around the carrier in group 3 (BoneCeramic(r) + 0.1mg simvastatin) followed by group 2 (BoneCeramic(r)), and almost no bone was formed in the other groups. Group 3 was significantly different compared to group 2, and both groups were significantly different compared to the other groups., Conclusion: Simvastatin combined with BoneCeramic(r) induced significantly greater amounts of newly formed bone and has great potential for the healing of bone defects.

The potential of increasing bone mass and preventing fractures in osteoporosis using stem cell therapy is currently an area of intense focus. However, there are very little data available regarding the postfracture bony defect healing efficacy under osteoporotic conditions. This study aims to investigate whether critical-sized segmental bone defects in a rabbit model of osteoporosis could be repaired using an allogenic stem cell-based tissue engineering (TE) approach and to investigate the potential influence of osteoporosis on the treatment efficacy. Rabbit fetal bone marrow mesenchymal stem cells (BMSCs) were harvested and expanded in vitro. Decalcified bone matrix (DBM) scaffolds were then seeded with allogenic fetal BMSCs and cultivated in osteogenic media to engineer BMSC/DBM constructs. Critical-sized radial defects were created in ovariectomized (OVX) rabbits and the defects were repaired either by insertion of BMSC/DBM constructs or by DBM scaffolds alone. Also, nonovariectomized age-matched (non-OVX) rabbits were served as control. At 3 months post-treatment under the osteoporotic condition (OVX rabbits), the BMSC/DBM constructs inserted within the defect generated significantly more bone tissue when compared to the DBM scaffold as demonstrated by the X-ray, microcomputed tomography, and histological analyses. In addition, when compared to a normal nonosteoporotic condition (age-matched non-OVX rabbits), the defect treatment efficacy was adversely affected by the osteoporotic condition with significantly less bone regeneration. This study demonstrated the potential of allogenic fetal BMSC-based TE strategy for repairing bone defects in an osteoporotic condition. However, the treatment efficacy could be considerably compromised in the OVX animals. Therefore, a more sophisticated strategy that addresses the complicated pathogenic conditions associated with osteoporosis is needed.

Purpose: Peripheral blood (PB) admixture should be minimized during numerical and functional, as well as cytokinetic analysis of bone marrow (BM) aspirates for research purposes. Therefore, purity assessment of the BM aspirate should be performed in advance. We investigated whether bone matrix vesicle (BMV)-bound bone alkaline phosphatase (ALP) could serve as a marker for the purity of BM aspirates., Results: Total ALP activity was significantly higher in BM serum (97 (176-124)U/L, median (range)) compared to PB serum (63 (52-73)U/L, p < 0.001). Agarose gel electrophoresis showed a unique bone ALP fraction in BM, which was absent in PB. Native polyacrylamide gel electrophoresis revealed the high molecular weight of this fraction, corresponding with membrane-bound ALP from bone matrix vesicles (BMV), as evidenced by electron microscopy. A serial PB admixture experiment of bone cylinder supernatant samples, rich in BMV-bound ALP, confirmed the sensitivity of this proposed quality assessment method. Furthermore, a BMV ALP fraction of ≥ 15% is suggested as cut-off value for minimal BM quality. Moreover, the BM purity declines rapidly with larger aspirated BM volumes., Conclusion: The exclusive presence of BMV-bound ALP in BM could serve as a novel marker to assess purity of BM aspirates., (Copyright © 2015. Published by Elsevier B.V.)

Objectives: To investigate whether a combination of demineralized bone matrix (DBM) and bone marrow mesenchymal stem cells (BMSCs) infected with adenovirus-mediated- bone morphogenetic protein (Ad-BMP-2) and transforming growth factor-β3 (Ad-TGF-β3) promotes the repair of the full-thickness cartilage lesions in pig model., Methods: BMSCs isolated from pig were cultured and infected with Ad-BMP-2(B group), Ad-TGF-β3 (T group), Ad-BMP-2 + Ad-TGF-β3(BT group), cells infected with empty Ad served as a negative group(N group), the expression of the BMP-2 and TGF-β3 were confirmed by immunofluorescence, PCR, and ELISA, the expression of SOX-9, type II collagen(COL-2A), aggrecan (ACAN) in each group were evaluated by real-time PCR at 1w, 2w, 3w, respectively. The chondrogenic differentiation of BMSCs was evaluated by type II collagen at 21d with immunohistochemical staining. The third-passage BMSCs infected with Ad-BMP-2 and Ad-TGF-β3 were suspended and cultured with DBM for 6 days to construct a new type of tissue engineering scaffold to repair full-thickness cartilage lesions in the femur condyles of pig knee, the regenerated tissue was evaluated at 1,2 and 3 months after surgery by gross appearance, H&E, safranin O staining and O'driscoll score., Results: Ad-BMP-2 and Ad-TGF-β3 (BT group) infected cells acquired strong type II collagen staining compared with Ad-BMP-2 (B group) and Ad-TGF-β3 (T group) along. The Ad-BMP-2 and Ad-TGF-β3 infected BMSCs adhered and propagated well in DBM and the new type of tissue engineering scaffold produced hyaline cartilage morphology containing a stronger type II collagen and safranin O staining, the O'driscoll score was higher than other groups., Conclusions: The DBM compound with Ad-BMP-2 and Ad-TGF-β3 infected BMSCs scaffold has a good biocompatibility and could well induce cartilage regeneration to repair the defects of joint cartilage. This technology may be efficiently employed for cartilage lesions repair in vivo.