3D-Printed Demineralized Bone Matrix-Based Conductive Scaffolds Combined with Electrical Stimulation for Bone Tissue Engineering Applications.

Bone is remodeled through a dynamic process facilitated by biophysical cues that support cellular signaling. In healthy bone, signaling pathways are regulated by cells and the extracellular matrix and transmitted via electrical synapses. To this end, combining electrical stimulation (ES) with conductive scaffolding is a promising approach for repairing damaged bone tissue. Therefore, "smart" biomaterials that can provide multifunctionality and facilitate the transfer of electrical cues directly to cells have become increasingly more studied in bone tissue engineering. Herein, 3D-printed electrically conductive composite scaffolds consisting of demineralized bone matrix (DBM) and polycaprolactone (PCL), in combination with ES, for bone regeneration were evaluated for the first time. The conductive composite scaffolds were fabricated and characterized by evaluating mechanical, surface, and electrical properties. The DBM/PCL composites exhibited a higher compressive modulus (107.2 MPa) than that of pristine PCL (62.02 MPa), as well as improved surface properties (i.e., roughness). Scaffold electrical properties were also tuned, with sheet resistance values as low as 4.77 × 105 Ω/sq for our experimental coating of the highest dilution (i.e., 20%). Furthermore, the biocompatibility and osteogenic potential of the conductive composite scaffolds were tested using human mesenchymal stromal cells (hMSCs) both with and without exogenous ES (100 mV/mm for 5 min/day four times/week). In conjunction with ES, the osteogenic differentiation of hMSCs grown on conductive DBM/PCL composite scaffolds was significantly enhanced when compared to those cultured on PCL-only and nonconductive DBM/PCL control scaffolds, as determined through xylenol orange mineral staining and osteogenic protein analysis. Overall, these promising results suggest the potential of this approach for the development of biomimetic hybrid scaffolds for bone tissue engineering applications.

Estrogen deficiency induces changes in bone matrix bound water that do not closely correspond with bone turnover.

Postmenopausal osteoporosis, marked by estrogen deficiency, is a major contributor to osteoporotic fractures, yet early prediction of fractures in this population remains challenging. Our goal was to explore the temporal changes in bone-specific inflammation, oxidative stress, bone turnover, and bone-matrix water, and their relationship with estrogen deficiency-induced modifications in bone structure and mechanical properties. Additionally, we sought to determine if emerging clinically translatable imaging techniques could capture early bone modifications prior to standard clinical imaging. Two-month-old female Sprague Dawley rats (n = 48) underwent ovariectomy (OVX, n = 24) or sham operations (n = 24). A subgroup of n = 8 rats per group was sacrificed at 2-, 5-, and 10-weeks post-surgery to assess the temporal relationships of inflammation, oxidative stress, bone turnover, bone matrix water, mechanics, and imaging outcomes. OVX rats exhibited higher body weight compared to sham rats at all time points. By 5-weeks, OVX animals showed elevated markers of inflammation and oxidative stress in cortical bone, which persisted throughout the study, while cortical bone formation rate did not differ from sham until 10-weeks. DXA outcomes did not reveal differences between OVX and sham at any time point. Bound water, assessed using ultrashort echo time magnetic resonance imaging (UTE MRI), was lower in OVX at the earliest time point (2-weeks) and reduced again at 10-weeks with no difference at 5-weeks. These data demonstrate that bound water assessment using novel UTE MRI technology was lower at the earliest time point following OVX. However, no temporal relationship with bone turnover, inflammation, or oxidative stress was observed at the time points assessed in this study. These findings underscore both the increased need to understand bone hydration changes and highlight the usefulness of UTE MRI for non-invasive bone hydration measurements.

CAD/CAM Titanium Meshes for GBR: A Case Series with Preliminary Histologic Analysis.

Titanium has been proposed as a mesh material for guided bone regeneration (GBR) since the 1990s. To overcome difficulties in shaping and adapting meshes to the defect, digital techniques were introduced to digitally print meshes capable of fitting the bone perfectly, reproduced through the patient's CT scan. Five patients were included in this case series, and their CBCT data were acquired and sent to the producer of the titanium meshes. 3D regenerative surgery was performed with titanium meshes and a mix of demineralized bovine bone matrix (DBBM) and autogenous bone (1:1 ratio). Radiographic measures were evaluated on paraxial sections of the CBCT through a dedicated software. When possible, regenerated bone samples were obtained at implant insertion. Four out of five regenerated areas healed without local or systemic complications. One mesh was removed after 2 months and 2 weeks due to exposure. The mean vertical bone gain was 4.3 ± 1.5 mm (range: 2.5 to 7 mm). Two histologic samples were obtained. In sample 1, bone tissue area and graft material area were 44.4% and 12.5%, respectively; in sample 2, the same parameters were 15.6% and 16.9%, respectively.

Functions and Metabolism of Amino Acids in Bones and Joints of Cats and Dogs.

The bone is a large and complex organ (12-15% of body weight) consisting of specialized connective tissues (bone matrix and bone marrow), whereas joints are composed of cartilage, tendons, ligaments, synovial joint capsules and membranes, and a synovial joint cavity filled with synovial fluid. Maintaining healthy bones and joints is a dynamic and complex process, as bone deposition (formation of new bone materials) and resorption (breakdown of the bone matrix to release calcium and phosphorus) are the continuous processes to determine bone balance. Bones are required for locomotion, protection of internal organs, and have endocrine functions to maintain mineral homeostasis. Joints are responsible for resisting mechanical stress/trauma, aiding in locomotion, and supporting the overall musculoskeletal system. Amino acids have multiple regulatory, compositional, metabolic, and functional roles in maintaining the health of bones and joints. Their disorders are prevalent in mammals and significantly reduce the quality of life. These abnormalities in companion animals, specifically cats and dogs, commonly lead to elective euthanasia due to the poor quality of life. Multiple disorders of bones and joints result from genetic predisposition and are heritable, but other factors such as nutrition, growth rate, trauma, and physical activity affect how the disorder manifests. Treatments for cats and dogs are primarily to slow the progression of these disorders and assist in pain management. Therapeutic supplements such as Cosequin and formulated diets rich in amino acids are used commonly as treatments for companion animals to reduce pain and slow the progression of those diseases. Also, amino acids (e.g., taurine, arginine, glycine, proline, and 4-hydroxyproline), and glucosamine reduce inflammation and pain in animals with bone and joint disorders. Gaining insight into how amino acids function in maintaining bone and joint health can aid in developing preventative diets and therapeutic supplementations of amino acids to improve the quality of life in companion animals.

The respective and dependent effects of scattering and bone matrix absorption on ultrasound attenuation in cortical bone.

Cortical bone is characterized by a dense solid matrix permeated by fluid-filled pores. Ultrasound scattering has potential for the non-invasive evaluation of changes in bone porosity. However, there is an incomplete understanding of the impact of ultrasonic absorption in the solid matrix on ultrasound scattering. In this study, maps were derived from scanning acoustic microscopy images of human femur cross-sections. Finite-difference time domain ultrasound scatter simulations were conducted on these maps. Pore density, diameter distribution of the pores, and nominal absorption values in the solid and fluid matrices were controlled. Ultrasound pulses with a central frequency of 8.2 MHz were propagated, both in through-transmission and backscattering configurations. From these data, the scattering, bone matrix absorption, and attenuation extinction lengths were calculated. The results demonstrated that as absorption in the solid matrix was varied, the scattering, absorption, and attenuation extinction lengths were significantly impacted. It was shown that for lower values of absorption in the solid matrix (less than 2 dB mm-1), attenuation due to scattering dominates, whereas at higher values of absorption (more than 2 dB mm-1), attenuation due to absorption dominates. This will impact how ultrasound attenuation and scattering parameters can be used to extract quantitative information on bone microstructure.

Influence of intramedullary pressure on Lacuno-Canalicular fluid flow: A systematic review.

While most of current models investigating bone remodelling are based on matrix deformation, intramedullary pressure also plays a role. Bone remodelling is orchestrated by the Lacuno-Canalicular Network (LCN) fluid-flow. The aim of this review was hence to assess the influence of intramedullary pressure on the fluid circulation within the LCN. Three databases (Science Direct, Web of Science, and PubMed) were used. The first phase of the search returned 731 articles, of which 9 respected the inclusion/exclusion criteria and were included. These studies confirm the association between intramedullary pressure and fluid dynamics in the LCN. Among the included studies, 7 experimental studies using animal models and 2 numerical models were found. The studies were then ranked according to the nature of the applied loading, either axial compression or direct cyclic intramedullary pressure. The current review revealed that there is an influence of intramedullary pressure on LCN fluid dynamics and that this influence depends on the magnitude and the frequency of the applied pressure. Two studies confirmed that the influence was effective even without bone matrix deformation. While intramedullary pressure is closely associated with LCN fluid, there is a severe lack of studies on this topic. STATEMENT OF SIGNIFICANCE: Since the 1990's, numerical models developed to investigate fluid flow in bone submicrometric porous network are based on the flow induced by matrix deformation. Bone fluid flow is known to be involved in cells stimulation and hence directly influences bone remodeling. Different studies have shown that intramedullary pressure is also associated with bone mechanosensitive adaptation. This pressure is developed in bone due to blood circulation and is increased during loading or muscle stimulation. The current article reviews the studies investigating the influence of this pressure on bone porous fluid flow. They show that fluid flow is involved by this pressure even without bone matrix deformation. The current review article highlights the severe lack of studies about this mechanism.

The Effect of Structural Characteristics of Deproteinized Spongy Bone on Activity of Adipose Tissue Mesenchymal Stromal Cells.

We studied the effect of structural properties of deproteinized spongy bone (DSB) on functional activity of adipose tissue mesenchymal stromal cells of (MSC) for the potential use of these materials as components of a combined tissue-engineered construct. The porosity of the structure of DSB samples and the pore size promote MSC adhesion, migration, and proliferation on their surface and in the depth, revealing the architectonics of this bone matrix. The depth of cell penetration into the samples (from 273 to 702 µm) and an increase in the total number of cells (from 302 on day 1 to 1744 on day 7) demonstrated MSC adhesion, migration, and proliferation. The viability of cultured MSC was preserved for up to 7 days. The obtained results prove the possibility of using allogeneic DSB from femoral heads as a bone matrix in tissue-engineered constructs in combination with MSC. Such constructs can be used to efficiently restore the structural and functional integrity of the bone tissue in abnormal processes of various etiopathogenesis associated with the formation of bone defects or bone tissue deficiency.

Demineralized bone matrix for repair and regeneration of maxillofacial defects: A narrative review.

OBJECTIVES: Demineralized bone matrix (DBM) is a well-established bone graft material widely accepted by dentists and the public for its favorable osteoconductivity and osteoinductive potential. This article aimed to provide a narrative review of the current therapeutic applications and limitations of DBM in maxillofacial bone defects. STUDY SELECTION, DATA, AND SOURCES: Randomized controlled trials, prospective or retrospective clinical studies, case series and reports, and systematic reviews. MEDLINE, PubMed, and Google Scholar were searched using keywords. CONCLUSIONS: Some evidence supported the therapeutic application of DBM in periodontal intrabony defects, maxillary sinus lifts, ridge preservation, ridge augmentation, alveolar cleft repair, orthognathic surgery, and other regional maxillofacial bone defects. However, the limitations of DBM should be considered when using it, including potential low immunogenicity, instability of osteoinductive potential, handling of the graft material, and patient acceptance. CLINICAL SIGNIFICANCE: With the increasing demand for the treatment of maxillofacial bone defects, DBM is likely to play a greater role as a promising bone graft material. Safe and effective combination treatment strategies and how to maintain a stable osteoinductive potential will be the future challenges of DBM research.

Preparation of black phosphorus@sodium alginate microspheres with bone matrix vesicle structure via electrospraying for bone regeneration.

Bone matrix vesicles are commonly acknowledged as the primary site of biomineralization in human skeletal tissue. Black phosphorus has exhibited favorable properties across various chemical and physical domains. In this investigation, a novel composite microsphere was synthesized through the amalgamation of sodium alginate (ALG) with black phosphorus nanosheets (BP) utilizing the electrospray (ES) technique. These microspheres were tailored to mimic the regulatory function of matrix vesicles (MV) upon exposure to a biomimetic mineralization fluid (SBF) during the biomineralization process. Results revealed that black phosphorus nanosheets facilitated the generation of hydroxyapatite (HA) on the microsphere surface. Live-dead assays and cell proliferation experiments showcased a cell survival rate exceeding 85 %. Moreover, wound healing assessments unveiled that M-ALG-BP microspheres exhibited superior migration capacity, with a migration rate surpassing 50 %. Furthermore, after 7 days of osteogenic induction, M-ALG-BP microspheres notably stimulated osteoblast differentiation. Particularly noteworthy, M-ALG-BP microspheres significantly enhanced osteogenic differentiation of osteoblasts and induced collagen production in vitro. Additionally, experiments involving microsphere implantation into mouse skeletal muscle demonstrated the potential for ectopic mineralization by ALG-BP microspheres. This investigation underscores the outstanding mineralization properties of ALG-BP microspheres and their promising clinical prospects in bone tissue engineering.

Mechanical insights into jawbone characteristics under chronic kidney disease: A comprehensive nanoindentation approach.

The mechanical properties of the jawbone play a critical role in determining the successful integration of dental prostheses. Chronic kidney disease (CKD) has been identified to abnormally accelerate bone turnover rates. However, the impact of CKD on the mechanical characteristics of the jawbone has not been extensively studied. This study sought to evaluate the time-dependent viscoelastic behaviors of rat jawbones, particularly in the scenarios both with and without CKD. We hypothesized that CKD might compromise the bone's innate toughening mechanisms, potentially owing to the time-dependent viscoelasticity of the bone matrix proteins. The maxillary and mandibular bones of Wistar rats were subjected to nanoindentation and Raman micro-spectroscopy. Load-hold-displacement curves from the cortical regions were obtained via nanoindentation and were mathematically characterized using a suitable viscoelastic constitutive model. Raman micro-spectroscopy was employed to identify nuanced vibrational changes in local molecular structures induced by CKD. The time course of indenter penetration onto cortical bones during the holding stage (creep behavior) can be mathematically represented by a series arrangement of the Kelvin-Voigt bodies. This configuration dictates the overall viscoelastic response observed during nanoindentation tests. The CKD model exhibited a reduced extent of viscoelastic contributions, especially during the initial ramp loading phase in both the maxillary and mandibular cortical bones. The generalized Kelvin-Voigt model comprises 2 K-Voigt elements that signify an immediate short retardation time (τ1) and a subsequent prolonged retardation time (τ2), respectively. Notably, the mandibular CKD model led to an increase in the delayed τ2 alongside an increase in non-enzymatic collagen cross-linking. These suggest that, over time, CKD diminishes the bone's capability for supplementary energy absorption and dimensional recovery, thus heightening their susceptibility to fractures.

Static magnetic field enhances the bone remodelling capacity of human demineralized bone matrix in a rat animal model of cranial bone defects.

The regeneration of bone defects that exceed 2 cm is a challenge for the human body, necessitating interventional therapies. Demineralized bone matrices (DBM) derived from biological tissues have been employed for bone regeneration and possess notable osteoinductive and osteoconductive characteristics. Nevertheless, their efficiency in regenerating critically sized injuries is limited, and therefore additional signaling cues are required. Thanks to the piezoelectric properties of the bone, external physical stimulation is shown to accelerate tissue healing. We have implanted human DBM in critically sized cranial bone defects in rat animal models and exposed them to an external magnetic field (1 T) to enhance endogenous bone formation. Our in vitro experiments showed the superior cytocompatibility of DBM compared to cell culture plates. Furthermore, alkaline phosphatase activity after 14 days and Alizarin red staining at 28 days demonstrated differentiation of rat bone marrow mesenchymal stem cells into bone lineage on DBM. Computer tomography images together with histological analyses showed that implanting DBM in the injured rats significantly enhanced bone regeneration. Notably, combining DBM transplantation with a 2 h daily exposure to a 1 T magnetic field for 2 weeks (day 7 to 21 post-surgery) significantly improved bone regeneration compared to DBM transplantation alone. This research indicates that utilizing external magnetic stimulation significantly enhances the potential of bone allografts to regenerate critically sized bone defects.

Variability of BMP-2 content in DBM products derived from different long bone.

Demineralized bone matrix (DBM) has been regarded as an ideal bone substitute as a native carrier of bone morphogenetic proteins (BMPs) and other growth factors. However, the osteoinductive properties diverse in different DBM products. We speculate that the harvest origin further contributing to variability of BMPs contents in DBM products besides the process technology. In the study, the cortical bone of femur, tibia, humerus, and ulna from a signal donor were prepared and followed demineralizd into DBM products. Proteins in bone martix were extracted using guanidine-HCl and collagenase, respectively, and BMP-2 content was detected by sandwich enzyme-linked immunosorbent assay (ELISA). Variability of BMP-2 content was found in 4 different DBM products. By guanidine-HCl extraction, the average concentration in DBMs harvested from ulna, humerus, tibia, and femur were 0.613 ± 0.053, 0.848 ± 0.051, 3.293 ± 0.268, and 21.763 ± 0.344, respectively (p < 0.05), while using collagenase, the levels were 0.089 ± 0.004, 0.097 ± 0.004, 0.330 ± 0.012, and 1.562 ± 0.008, respectively (p < 0.05). In general, the content of BMP-2 in long bones of Lower limb was higher than that in long bones of upper limb, and GuHCl had remarkably superior extracted efficiency for BMP-2 compared to collagenase. The results suggest that the origin of cortical bones harvested to fabricate DBM products contribute to the variability of native BMP-2 content, while the protein extracted method only changes the measured values of BMP-2.

Numerical calculation of streaming potential around osteocytes under human gait loading.

Streaming potential is a type of stress-generated potential in bone that affects the electrical environment of osteocytes and may play a role in bone remodeling. Because the electrical environment around osteocytes has been difficult to measure experimentally until now, a numerical solid-liquid-streaming potential coupling method was proposed to analyze the streaming potential generated by bone deformation in the lacunae and canaliculus network (LCN) of the bone. Using this method, the cellular shear stress caused by liquid flow on the osteocyte surface was first calculated, and the results were consistent with those reported in the literature. Subsequently, the streaming potentials in the LCN caused by bone matrix deformation under an external gait load were calculated numerically. The results showed that the streaming potential increased slowly in the lacuna and relatively rapidly in the canaliculus and that the streaming potential increased with a decrease in the radius or an increase in the length of the canaliculus. The results also showed that relatively large gaps between the lacunae and osteocytes could induce higher streaming potentials under the same loading.

A physicochemical evaluation of ossein-hydroxyapatite within the bovine bone matrix revealed demineralization and making type I collagen available as a result of processing and solubilization by acids.

Bovine bone is an animal-origin matrix rich in type I collagen (COL I) and it necessitates prior demineralization and makes COL I available. This study investigated the ossein-hydroxyapatite physicochemical properties evaluation as a result of processing and solubilization by acids and revealed the bone matrix demineralization and making COL I available. The tibia residue from bovine sources was processed, ground, and transformed into bone matrix powder. The bone matrix was solubilized in acetic acid followed by lactic acid. The bone matrix was evaluated as a result of processing and solubilization by acids: ossein and hydroxyapatite percentages by nitrogen and ash content, mineral content, particle size distribution, Fourier-transformation infrared spectroscopy, x-ray diffraction, and scanning electron microscope. For the obtained residual extracts, pH and mineral content were evaluated. The solubilization by acids affected the ossein-hydroxyapatite physicochemical properties, and the bone matrix solubilized by acetic and lactic acid showed the preservation of the ossein alongside the loss of hydroxyapatite. The processing and the solubilization by acids were revealed to be a  alternative to bone matrix demineralization and enabling the accessibility of bone COL I. PRACTICAL APPLICATION: Bovine bone is an abundant type I collagen source, but processing maneuvers and demineralization effect present limitations due to the rigidity of the structural components. Exploring methodologies to process and demineralize will allow type I collagen to be obtained from the bone source, and direct and amplify the potentialities in the chemical and food industries. The research focused on bone sources and collagen availability holds paramount significance, and promotes repurposing agribusiness residues and development of protein-base products.

Comparison of different bone substitutes in the repair of rat calvaria critical size defects: questioning the need for alveolar ridge presentation.

OBJECTIVE: This study aimed to evaluate the effectiveness of biomaterials in bone healing of critical bone defects created by piezoelectric surgery in rat calvaria. METHOD AND MATERIALS: Histomorphologic analysis was performed to assess bone regeneration and tissue response. Fifty animals were randomized into five groups with one of the following treatments: Control group (n = 10), spontaneous blood clot formation with no bone fill; BO group (Bio-Oss, Geistlich Pharma; n = 10), defects were filled with bovine medullary bone substitute; BF group (Bonefill, Bionnovation; n = 10), defects were filled with bovine cortical bone substitute; hydroxyapatite group (n = 10), defects were filled with hydroxyapatite; calcium sulfate group (n = 10), defects were filled with calcium sulfate. Five animals from each group were euthanized at 30 and 45 days. The histomorphometry calculated the percentage of the new bone formation in the bone defect. RESULTS: All data obtained were evaluated statistically considering P < .05 as statistically significant. The results demonstrated the potential of all biomaterials for enhancing bone regeneration. The findings showed no statistical differences between all the biomaterials at 30 and 45 days including the control group without bone grafting. CONCLUSION: In conclusion, the tested biomaterials presented an estimated capacity of osteoconduction, statistically nonsignificant between them. In addition, the selection of biomaterial should consider the specific clinical aspect, resorption rates, size of the particle, and desired bone healing responses. It is important to emphasize that in some cases, using no bone filler might provide comparable results with reduced cost and possible complications questioning the very frequent use of ridge presentation procedures.

Comparison of Fusion Rates among Various Demineralized Bone Matrices in Posterior Lumbar Interbody Fusion.

Background and Objectives: Posterior lumbar interbody fusion (PLIF) plays a crucial role in addressing various spinal disorders. The success of PLIF is contingent upon achieving bone fusion, as failure can lead to adverse clinical outcomes. Demineralized bone matrix (DBM) has emerged as a promising solution for promoting fusion due to its unique combination of osteoinductive and osteoconductive properties. This study aims to compare the effectiveness of three distinct DBMs (Exfuse®, Bongener®, and Bonfuse®) in achieving fusion rates in PLIF surgery. Materials and Methods: A retrospective review was conducted on 236 consecutive patients undergoing PLIF between September 2016 and February 2019. Patients over 50 years old with degenerative lumbar disease, receiving DBM, and following up for more than 12 months after surgery were included. Fusion was evaluated using the Bridwell grading system. Bridwell grades 1 and 2 were defined as 'fusion', while grades 3 and 4 were considered 'non-fusion.' Clinical outcomes were assessed using visual analog scale (VAS) scores for pain, the Oswestry disability index (ODI), and the European quality of life-5 (EQ-5D). Results: Fusion rates were 88.3% for Exfuse, 94.3% for Bongener, and 87.7% for Bonfuse, with no significant differences. All groups exhibited significant improvement in clinical outcomes at 12 months after surgery, but no significant differences were observed among the three groups. Conclusions: There were no significant differences in fusion rates and clinical outcomes among Exfuse, Bongener, and Bonfuse in PLIF surgery.

Variables Influencing Bone Formation After Transcrestal Sinus Floor Elevation: Radiographic and Tomographic Evaluations.

PURPOSE: To evaluate the influence of initial implant protrusion within the subantral space on hard tissue gain for implants placed simultaneously with transcrestal sinus floor elevation (TSFE) with a biomaterial. MATERIALS AND METHODS: A total of 50 implants were placed after TSFE in 44 patients using either a human demineralized bone matrix or a deproteinized bone mineral matrix. Intraoral radiographs were obtained before and immediately after surgery. CBCT scans were obtained at the last follow-up (mean: 6.6 years). RESULTS: The initial bone crest height was 4.6 ± 1.4 mm, and the initial protrusion of the implants above the sinus floor was 3.5 ± 1.4 mm. At the follow-up assessments, the hard tissue mean gain was 2.5 ± 1.5 mm, resulting in a mean residual protrusion of 1.1 ± 1.3 mm. Only 10 implants did not protrude above the apical level of hard tissue. Positive correlations were found between hard tissue gain and initial protrusion (r = 0.55; 95% CI: 0.32 to 0.72; P = .0001), between the initial and final protrusions (r = 0.38; 95% CI: 0.10 to 0.60; P = .007), and between the follow-up period and final protrusion (r = 0.35; 95% CI: 0.07 to 0.58; P = .012). CONCLUSIONS: The higher the initial protrusion was, the higher were the hard tissue gain and the final protrusion of the implant above the apical level of the hard tissue.

Possible involvement of HtrA1 serine protease in the onset of osteoporotic bone extracellular matrix changes.

High-temperature requirement A1 (HtrA1), a multidomain serine protease acting on Extracellular matrix (ECM) rearrangement, is also secreted by osteoblasts and osteoclasts. Recent and conflicting literature highlights HtrA1's role as a controller of bone remodeling, proposing it as a possible target for pathologies with unbalanced bone resorption, like Osteoporosis (OP). To add knowledge on this molecule function in bone physiopathology, here we compared HtrA1 distribution in the ECM of healthy (H) and OP bone tissue, also examining its localization in the sites of new bone formation. HtrA1 was homogeneously expressed in the mature bone ECM of H tissue showing a 55.6 ± 16.4% of the stained area, with a significant (p=0.0001) decrease in OP percentage stained area (21.1 ± 13.1). Moreover, HtrA1 was present in the endosteum and cells involved in osteogenesis, mainly in those "entrapped" in woven bone, whereas osteocytes in mature lamellar bone were negative. Based on our previous observation in OP tissue of a significantly increased expression of Decorin and Osteocalcin, both involved in bone mineralization and remodeling and equally substrates for HtrA1, we speculate that HtrA1 by controlling the proper amount of Decorin and Osteocalcin favors normal bone maturation and mineralization. Besides, we suggest that late-osteoblasts and pre-osteocytes secrete HtrA1 in the adjacent matrix whilst proceeding with their maturation and that HtrA1 expression is further modified during the remodeling from woven to the lamellar bone. Overall, our data suggest HtrA1 as a positive regulator of bone matrix formation and maturation: its reduced expression in mature OP bone, affecting protein content and distribution, could hamper correct bone remodeling and mineralization.

Self-Assembled Nanocomposite Hydrogels as Carriers for Demineralized Bone Matrix Particles and Enhanced Bone Repair.

Demineralized bone matrix (DBM) has been widely used as an allogeneic alternative to autologous bone graft for bone repair. However, more extensive use of DBM is limited due to its particulate nature after demineralization and rapid particle dispersion following irrigation, resulting in unpredictable osteoinductivity. Here, a new design of injectable hydrogel carriers for DBM that combine self-healing ability and osteogenic properties based on the self-assembly of guanidinylated hyaluronic acid and silica-rich nanoclays is reported. The nanoclays serve as reversible linkages to form a dynamic hydrogel network with the guanidine moieties on the polymer chains. Gelation kinetics and mechanical properties can be controlled by altering nanoclay content in the hydrogel. The resulting hydrogel exerts self-healing ability due to its dynamic crosslinks and well retains its overall performance with high DBM loading. The hydrogel exhibits great cytocompatibility and osteogenic effects mediated by the nanoclays. In vivo delivery of DBM using the nanocomposite hydrogel further demonstrates robust bone regeneration in a mouse calvarial defect model in comparison to DBM delivered with aqueous HA. This work suggests a promising hydrogel platform for many applications including therapeutic delivery and tissue engineering.

Augmented drug resistance of osteosarcoma cells within decalcified bone matrix scaffold: The role of glutamine metabolism.

Due to the lack of a precise in vitro model that can mimic the nature microenvironment in osteosarcoma, the understanding of its resistance to chemical drugs remains limited. Here, we report a novel three-dimensional model of osteosarcoma constructed by seeding tumor cells (MG-63 and MNNG/HOS Cl no. 5) within demineralized bone matrix scaffolds. Demineralized bone matrix scaffolds retain the original components of the natural bone matrix (hydroxyapatite and collagen type I), and possess good biocompatibility allowing osteosarcoma cells to proliferate and aggregate into clusters within the pores. Growing within the scaffold conferred elevated resistance to doxorubicin on MG-63 and MNNG/HOS Cl no. 5 cell lines as compared to two-dimensional cultures. Transcriptomic analysis showed an increased enrichment for drug resistance genes along with enhanced glutamine metabolism in osteosarcoma cells in demineralized bone matrix scaffolds. Inhibition of glutamine metabolism resulted in a decrease in drug resistance of osteosarcoma, which could be restored by α-ketoglutarate supplementation. Overall, our study suggests that microenvironmental cues in demineralized bone matrix scaffolds can enhance osteosarcoma drug responses and that targeting glutamine metabolism may be a strategy for treating osteosarcoma drug resistance.