Human Bone Matrix Research Articles

Converting ocean nacre into bone mineral matrix composite for bone regeneration- in vitro and in vivo studies

Nacre of Pinctada maxima is a natural biomineralized matrix, appeared 7 million years before hominins. In this study, we converted nacre into a self-setting particle bound with multiple calcium orthophosphates that reassemble mammals’ bone mineral matrices for induction of bone regeneration. The nacre-based calcium orthophosphates composite (NCOC) exhibited a compression strength of 10 MPa, which is superior to human trabecular bone. In vitro bioactivity tests revealed the formation of apatite with nano-porous flake-like crystals on the composite surface that mimic HA structure of a human bone matrix. NCOC demonstrated efficient attachment and proliferation of osteoblast cells, promoting osteogenic differentiation by increasing expressions of RUNX2 and OPN. In vivo studies using rabbit back fascia demonstrated that NCOC displays better bone healing and biocompatibility than conventional bone substitute apatite in critical bone defect models. The degradation of calcium carbonate crystal in vivo does not compromise structural integrity of NCOC. Overall, our data showed that NCOC produced through self-setting reactions, presents advantages such as accelerated biodegradation and osteostimulative properties, making it a promising bone substitute for effective bone regeneration.

Recyclable thermo-responsive elastin-based adhesives with tough underwater adhesion and rapid hemostasis ability

Protein-based underwater adhesives have attracted tremendous attention due to their excellent biocompatibility compared to adhesives prepared with synthetic polymers. However, the fabrication of protein-based adhesives always requires multiple successive steps, and their underwater adhesion is quite limited. Inspired by the human elastic fibers, bone matrix, and marine adhesive proteins, a thermo-responsive underwater adhesive was fabricated through a facial one-step complexation of elastin (ELN), hydroxyapatite (HAP), and tannic acid (TA) based on multiple non-covalent molecular interactions. During underwater adhesion, the adhesive can perform sufficient flowability to adhere seamlessly and strongly to the substrates upon heating at 37 ℃ and then significantly increase its cohesion through cooling down to 25 ℃ to sustain adhesion. The regulation of the balance between adhesion and cohesion in response to temperature change enables the adhesive to exhibit a robust underwater adhesion ranging from 0.3 to 1.1 MPa to the substrates with low, medium, or high surface energy, outperforming most protein-based adhesives. Importantly, the adhesive can be further processed into powders with well-maintained underwater adhesion by a simple drying and grinding method, facilitating adhesion onto irregular-shaped target sites, reusability, and closed-loop recycling. The adhesive powder also possesses a rapid in vivo hemostatic property, suggesting a potential biomedical application.

Abstract PR002: Ferroptosis inducers can antagonize prostate cancer-bone interactions

Abstract Prostate cancer (PCa) is a leading cause of mortality, primarily due to its ability to metastasize to the bone. The incidence, rates of metastasis and mortality rates are higher in men of African descent compared to other races. The transcription factor High Mobility Group AT-Hook 2 (HMGA2) plays a crucial role in regulating gene expression and has been implicated in tumorigenesis and the metastatic process. Our recent study revealed that overexpression of the wild-type/full-length HMGA2 isoform in PCa cells promotes cancer progression by triggering epithelial mesenchymal transition (EMT), whereas truncated HMGA2 isoform promotes progression through oxidative stress signaling. Ferroptosis is a regulated form of cell death induced by the accumulation of iron-dependent lipid reactive oxygen species (ROS). Exploiting this oxidative stress pathway offers a promising therapeutic approach for cancer treatment, especially for apoptosis-resistant cancer cells. In our investigation, we aimed to assess the effectiveness of RSL3, a known ferroptosis inducer, in inhibiting interactions between prostate cancer cells and bone. To achieve this, we co-cultured various human PCa cell lines: C4-2B (Caucasian/bone metastatic), C4-2B MDVR (enzalutamide-resistant), MDAPCa-2b (African American/bone metastatic), LNCaP (Caucasian/non-metastatic) that stably overexpresses Neo control, HMGA2 wild-type or truncated isoform with human ground bone matrix powder (GBM) to recapitulate the bone microenvironment. We collected the conditioned media (CM) from these co-cultures and subsequently added it to parental LNCaP cells, with or without RSL3 ferroptosis inducer, plus or minus Ferrostatin-1 (Fer-1) ferroptosis inhibitor. We then assessed cell viability, migration, and lipid ROS levels. Our results demonstrated that LNCaP cells treated with CM from cancer cells co-cultured with GBM exhibited increased cell viability and migration. However, the addition of RSL3 effectively inhibited cell viability by inactivating GPX4 antioxidant and triggering lipid ROS production; this could be reversed by Fer-1. This suggests that ferroptosis inducers such as RSL3 hold potential as a treatment strategy for prostate cancer bone metastasis. Further investigation is necessary to comprehensively evaluate the impact of RSL3 treatments and elucidate the underlying mechanisms involved. By exploring these avenues, we can advance our understanding and potentially develop targeted therapies to combat the devastating effects of prostate cancer bone metastasis. Acknowledgements: These studies were supported by NIH/NIGMS/RISE SR25GM060414 and NIH/NIMHD 2U54MD007590; 5U54MD013376. Citation Format: Precious Elechi Dike, Taaliah Campbell, Valerie Odero-Marah. Ferroptosis inducers can antagonize prostate cancer-bone interactions [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr PR002.

A Modified Technique for Lateral Column Lengthening as Part of Adult Acquired Flatfoot Deformity Correction: An Intact Medial Calcaneal Cortex, a Low-Profile Locking Plate With a Combination of Autologous Calcaneal Bone Graft and Demineralized Human Bone Matrix

Adult acquired flatfoot deformity encompasses deformities of the entire foot and ankle, with most clinical presentations comprising of a flexible planovalgus deformity and forefoot abduction. Numerous operative procedures have been proposed, and a lateral column–lengthening procedure is advocated to correct the forefoot abduction. Autologous iliac crest bone graft is routinely inserted into the opening-wedge osteotomy at the anterior calcaneum. Fixation methods are also variable with increasing use of locking plates to achieve stability at this osteotomy. Harvesting the iliac crest graft exposes the patient to significant donor site morbidity. We describe a modified lateral column–lengthening technique, leaving the medial calcaneum cortex intact, packing a combination of autologous calcaneal bone graft, and demineralized bone matrix into the osteotomy site, fixed with a low-profile locking plate. In the short term, our technique offers reduced rates of wound complications, symptomatic hardware, nonunion, and deformity recurrence when compared with published rates. Level of Evidence: Level IV, Case Series (retrospective).

Deproteinized Bone Tissue as a Matrix for Tissue-Engineered Construction: Experimental Study

Background. At present, for a number of reasons the complete bone defect replacement with autogenous bone is not always possible. Bone substitute materials are used as an alternative to autogenous bone tissue and can be of either biological or non-biological origin. One of the ways of development of reconstructive technologies is the use of tissue-engineered constructs that fully imitate autogenous bone tissue in the required volume.
 Aim of study to define in vivo the possibility of using deproteinized human cancellous bone tissue as a matrix for creating tissue-engineered constructs.
 Methods. An in vivo study was carried out on NZW rabbits. To create a construct, we used the fragments of deproteinized cancellous bone tissue of the human femoral head and stromal vascular fraction of rabbit adipose tissue as a matrix. Bone defect modeling with its subsequent replacement was performed to evaluate the efficacy of reparative osteogenesis during bone defects reconstruction. Study groups were defined: group 1 (control) surgical modeling of a bone defect of the femur without its reconstruction; group 2 surgical modeling of a bone defect of the femur with its reconstruction using fragments of deproteinized cancellous bone matrix; group 3 surgical modeling of a bone defect of the femur with its reconstruction using fragments of deproteinized cancellous bone matrix in combination with stromal vascular fraction of adipose tissue (according to ACP SVF technology).
 Results. Comparative analysis of reparative processes in case of applying tissue-engineered constructs based on deproteinized human cancellous bone matrix in combination with adipose tissue-derived stromal vascular fraction on in vivo experimental model revealed that the use of these bone substitute materials contributes not only to an early activation of reparative regeneration of main structural elements of the bone tissue in the area of the bone defect replacement, but also to its well-timed differentiation. This determines the restoration of structural and functional viability of the bone tissue at the damage site without developing discernible reactive inflammation. Moreover, the effect of the selected tissue-engineered construct with the combined influence of several factors (ACP SVF) in its composition turned out to be more effective in stimulating bone tissue repair and differentiation.
 Conclusion. Combination of SVF and deproteinized bone matrix for creating tissue-engineered constructs enables to engage several regeneration mechanisms and accelerate the process of bone defect replacement in comparison with isolated deproteinized bone matrix without bone defect reconstruction.

Is human bone matrix a sufficient augmentation method revising loosened pedicle screws in osteoporotic bone? – A biomechanical evaluation of primary stability

IntroductionDespite good screw anchorage and safe screw trajectory, screw loosening occurs in several cases, especially in osteoporotic individuals. The aim of this biomechanical analysis was to evaluate the primary stability of revision screw placement in individuals with reduced bone quality. Therefore, revision via enlarged diameter screws was compared to the use of human bone matrix as augmentation to improve the bone stock and screw coverage. Methods11 lumbar vertebral bodies from cadaveric specimens with a mean age of 85.7 years (± 12.0 years) at death were used. 6.5 mm diameter pedicle screws were inserted in both pedicles and hereafter loosened using a fatigue protocol. Screws were revised inserting a larger diameter screw (8.5 mm) in one pedicle and a same diameter screw with human bone matrix augmentation in the other pedicle. The previous loosening protocol was then reapplied, comparing maximum load and cycles to failure between both revision techniques. Insertional torque was continuously measured during insertion of both revision screws. FindingsThe number of cycles and the maximum load until failure were significantly greater in enlarged diameter screws than in augmented screws. The enlarged screws' insertional torque was also significantly higher than of the augmented screws. InterpretationHuman bone matrix augmentation does not reach the same ad-hoc fixation strength as enlarging the screw's diameter by 2 mm and is therefore biomechanically inferior. Regarding the immediate stability, a thicker screw should therefore be prioritised.

Abstract IA041: Basic science mechanisms associated with bone tumor microenvironment and health disparities in breast and prostate cancer

Abstract Prostate cancer (PCa) and breast cancer (BCa) are leading causes of cancer related deaths in American men and women, and morbidity is due to metastasis preferentially to bone. Compared to Caucasian American (CA), African American (AA) men and women have a higher bone density, as well as mortality rate due to bone metastasis. Recent studies have shown that high mobility group two (HMGA2), a non-histone chromatin binding protein, plays a critical role in promoting epithelial-mesenchymal transition (EMT) and metastasis. HMGA2 full-length/wild-type and truncated (without the 3’UTR) isoforms have been shown to be overexpressed in several cancers, however their distinct roles in metastasis have not been reported. Our laboratory focuses on tumor-micronenvironmental interactions, particularly, how PCa cells interact with bone at the metastatic site. We have previously published that PCa cells may degrade hydroxyapatite, the major inorganic component of bone, to release calcium which promotes paracrine cell proliferation and migration, and this signaling increases with increased bone density. We hypothesize that EMT is associated with prostate health disparities and that HMGA2 isoforms may play differential roles at the bone metastatic site. We examined EMT marker expression in breast and prostate patient tissue and cell lines by immunohistochemistry (IHC), RNA In Situ Hybridization (RISH), western blot, and real-time PCR. Next, LNCaP PCa cell line stably overexpressing either full length or truncated HMGA2 was co-cultured with low (100 mg) or high (200 mg) hydroxyapatite, demineralized bone matrix or human ground bone matrix, for 6 days followed by collection of condition media (CM). CM was added to parental LNCaP cells for various time points followed by analysis of paracrine cell proliferation using MTS assay, cell migration across collagen using boyden chamber, and the effect on various pathways using western blot analysis. EMT marker (Snail and Cathepsin L) protein expression was higher in AA normal and cancer tissue compared to CA, while it was intermediate in Bahamian tissue. Wild-type HMGA2 mRNA expression was higher in metastatic patient tissue compared to truncated HMGA2, and higher in AA compared to CA tissue. CM taken from LNCaP cells overexpressing wild-type and truncated HMGA2 co-cultured with hydroxyapatite, demineralized bone matrix or ground bone matrix when added to parental LNCaP cells led to higher paracrine cell proliferation and paracrine cell migration. This was associated with MAPK activation that could be abrogated by MAPK inhibitor, UO126. These results indicate that EMT is higher in AA compared to CA. Additionally, both wild-type and truncated HMGA2 interaction with bone matrix promotes paracrine cell proliferation and migration via ERK signaling. Our study shows that wild-type and truncated isoforms of HMGA2 mediate PCa/bone interactions which may lead to increased bone metastasis, particularly in African American men that have higher bone density. Citation Format: Taaliah Campbell, Ohuod Hawsawi, Bor-Jang Hwang, Liza J. Burton, Quentin Loyd, Veronica Henderson, Simone Howard, Camille Ragine, Robin Roberts, Andrew Gachii, Valerie Odero-Marah. Basic science mechanisms associated with bone tumor microenvironment and health disparities in breast and prostate cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA041.

Abstract C033: Investigating the differential roles of HMGA2 isoforms in the bone microenvironment

Abstract Prostate cancer (PCa) is the second leading cause of cancer related deaths in American men. Compared to Caucasian men, African American males have a higher bone density, as well as mortality rate due to bone metastasis. Recent studies have shown that High Mobility Group A2 (HMGA2), a non-histone chromatin binding protein, plays a critical role in promoting metastasis. HMGA2 full-length/wild-type and truncated (without the 3’UTR) isoforms have been shown to be overexpressed in several cancers, however their distinct roles in metastasis have not been reported. Our laboratory focuses on tumor-micronenvironmental interactions. We have previously published that PCa cells degrades hydroxyapatite, the major inorganic component of bone, to release calcium which promotes paracrine cell proliferation and migration, and this signaling increases with increased bone density. We hypothesize that HMGA2 isoforms may play differential roles at the bone metastatic site and effect paracrine cell proliferation and migration. LNCaP PCa cell line stably overexpressing either full length or truncated HMGA2 was co-cultured with low (100 mg) or high (200 mg) hydroxyapatite or human bone matrix powder, for 6 days followed by collection of conditioned media (CM). CM was co-cultured with parental LNCaP cells for various time points followed by analysis of several signaling pathways using western blot analysis, as well as analysis of paracrine cell proliferation using MTS assay, and cell migration across collagen using boyden chamber. Results reveal that co-culture of LNCaP cells expressing WT and TR HMGA2 with bone components has a paracrine effect on MAPK signaling. This was accompanied by a paracrine increase in cell migration but not cell proliferation with CM from truncated HMGA2-expressing cells co-cultured with bone, that can be abrogated by MAPK inhibition. Our study shows that wild-type and truncated isoforms of HMGA2 may differentially mediate PCa/hydroxyapatite bone interactions at the bone metastatic site, which may contribute to bone metastasis, particularly in African American men that have higher bone density. Acknowledgements: These studies were supported by NIH/NIGMS/RISE SR25GM060414 and NIH/NIMHD 2U54MD007590; 5U54MD013376-8281. Citation Format: Taaliah Campbell, Ohuod Hawsawi, Veronica Henderson, Valerie Odero-Marah. Investigating the differential roles of HMGA2 isoforms in the bone microenvironment [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C033.

Progress on carbon dots and hydroxyapatite based biocompatible luminescent nanomaterials for cancer theranostics

Despite the significant advancement in cancer diagnosis and therapy, a huge burden remains. Consequently, much research has been diverted on the development of multifunctional nanomaterials for improvement in conventional diagnosis and therapy. Luminescent nanomaterials offer a versatile platform for the development of such materials as their intrinsic photoluminescence (PL) property offers convergence of diagnosis as well as therapy at the same time. However, the clinical translation of nanomaterials faces various challenges, including biocompatibility and cost-effective scale up production. Thus, luminescent materials with facile synthesis approach along with intrinsic biocompatibility and anticancerous activity hold significant importance. As a result, carbon dots (CDs) and nanohydroxyapatite (nHA) have attracted much attention for the development of optical imaging probes. CDs are the newest members of the carbonaceous nanomaterials family that possess intrinsic luminescent and therapeutic properties, making them a promising candidate for cancer theranostic. Additionally, nHA is an excellent bioactive material due to its compositional similarity to the human bone matrix. The nHA crystal can efficiently host rare-earth elements to attain luminescent property, which can further be implemented for cancer theranostic applications. Herein, the development of CDs and nHA based nanomaterials as multifunctional agents for cancer has been briefly discussed. The emphasis has been given to different synthesis strategies leading to different morphologies and tunable PL spectra, followed by their diverse applications as biocompatible theranostic agents. Finally, the review has been summarized with the current challenges and future perspectives.

Regenerative medicine: characterization of human bone matrix gelatin (BMG) and folded platelet-rich fibrin (F-PRF) membranes alone and in combination (sticky bone)

During the last two decades autologous platelet and leukocyte rich products (PRP; PRF), opened new perspectives in regenerative medicine. In particular regenerative dentistry played a pioneer role in the application of these products in bone regenerative cases. Many aspects of cytokines, such as, growth factor release, blood cell content and its characterization were reported, but some practical questions are still unanswered in the preparation of PRF membranes and sticky bones. A new folding technique was introduced that created a good quality, pliable, and strong F-PRF membrane with a dense fibrin network and more homogenous blood cell distribution. F-PRF produced a very promising sticky bone combined with human freeze-dried cortical bone matrix gelatin (BMG). There hasn’t been much focus on the quality and character of the applied bone and the optimal membrane/bone particle ratio has not been reported. A 0.125 g BMG/ml plasma (1 g/8 ml) seems like the ideal combination with maximal BMG adhesion capacity of the membrane. Particle distribution of BMG showed that 3/4 of the particles ranged between 300–1000 µ, the remnant 1/4 was smaller than 300 µ. The whole F-PRF membrane and its parts were compared with conventional A-PRF membrane concerning their resistance against proteolytic digestion. The F-PRF was superior to A-PRF, which dissolved within 4–5 days, while F-PRF was destroyed only after 11 days, so this provides a better chance for local bone morphogenesis. The F-PRF pieces had similar resistance to the whole intact one, so they can be ideal for surgical procedures without risk of fast disintegration.

Subcanalicular Nanochannel Volume Is Inversely Correlated With Calcium Content in Human Cortical Bone.

The spatial distribution of mineralization density is an important signature of bone growth and remodeling processes, and its alterations are often related to disease. The extracellular matrix of some vertebrate mineralized tissues is known to be perfused by a lacunocanalicular network (LCN), a fluid-filled unmineralized structure that harbors osteocytes and their fine processes and transports extracellular fluid and its constituents. The current report provides evidence for structural and compositional heterogeneity at an even smaller, subcanalicular scale. The work reveals an extensive unmineralized three-dimensional (3D) network of nanochannels (~30 nm in diameter) penetrating the mineralized extracellular matrix of human femoral cortical bone and encompassing a greater volume fraction and surface area than these same parameters of the canaliculi comprising the LCN. The present study combines high-resolution focused ion beam-scanning electron microscopy (FIB-SEM) to investigate bone ultrastructure in 3D with quantitative backscattered electron imaging (qBEI) to estimate local bone mineral content. The presence of nanochannels has been found to impact qBEI measurements fundamentally, such that volume percentage (vol%) of nanochannels correlates inversely with weight percentage (wt%) of calcium. This mathematical relationship between nanochannel vol% and calcium wt% suggests that the nanochannels could potentially provide space for ion and small molecule transport throughout the bone matrix. Collectively, these data propose a reinterpretation of qBEI measurements, accounting for nanochannel presence in human bone tissue in addition to collagen and mineral. Further, the results yield insight into bone mineralization processes at the nanometer scale and present the possibility for a potential role of the nanochannel system in permitting ion and small molecule diffusion throughout the extracellular matrix. Such a possible function could thereby lead to the sequestration or occlusion of the ions and small molecules within the extracellular matrix. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Influence of Metastatic Bone Lesion Type and Tumor Origin on Human Vertebral Bone Architecture, Matrix Quality, and Mechanical Properties.

Metastatic spine disease is incurable, causing increased vertebral fracture risk and severe patient morbidity. Here, we demonstrate that osteolytic, osteosclerotic, and mixed bone metastasis uniquely modify human vertebral bone architecture and quality, affecting vertebral strength and stiffness. Multivariable analysis showed bone metastasis type dominates vertebral strength and stiffness changes, with neither age nor gender having an independent effect. In osteolytic vertebrae, bone architecture rarefaction, lower tissue mineral content and connectivity, and accumulation of advanced glycation end-products (AGEs) affected low vertebral strength and stiffness. In osteosclerotic vertebrae, high trabecular number and thickness but low AGEs, suggesting a high degree of bone remodeling, yielded high vertebral strength. Our study found that bone metastasis from prostate and breast primary cancers differentially impacted the osteosclerotic bone microenvironment, yielding altered bone architecture and accumulation of AGEs. These findings indicate that therapeutic approaches should target the restoration of bone structural integrity. © 2022 American Society for Bone and Mineral Research (ASBMR).

Duration-Dependent Increase of Human Bone Matrix Mineralization in Long-Term Bisphosphonate Users with Atypical Femur Fracture.

Bisphosphonates (BPs) are the most widely used drugs for the treatment of osteoporosis but prolonged use of BPs might increase the risk of atypical femur fracture (AFF). There are only a few studies that address the bone material quality in patients on long-term BP treatment with or without AFFs. We analyzed 52 trans-iliac bone biopsies from patients on long-term BP therapy with (n=26) and without (n=26) AFF. At the microscopic level, the degree of mineralization of bone (DMB) was assessed on whole bone by X-ray digitized microradiography while microhardness by Vickers microindentation, and bone matrix characteristics by Fourier transform infrared microspectroscopy (FTIRM) (mineral/organic ratio, mineral maturity and crystallinity, and collagen maturity) were measured at random focal areas. The AFF patients were treated longer than non-AFF patients (9.7 ± 3.3 years versus 7.9 ± 2.7 years). As expected, bone remodeling was low in both groups, without difference between them. The AFF group had significantly higher DMB in cortical bone (+2.9%, p=.001), which remained so after adjusting for treatment duration (p=.007), and showed a trend in cancellous bone (+1.6%, p=.05). Consistent with higher DMB, heterogeneity index (HI) was lower in the AFF than in the non-AFF group, illustrating lower heterogeneity of mineralization in the AFF group. A significant positive correlation between the duration of treatment and DMB in cortical bone was found in AFF, and not in the non-AFF group. Microhardness and bone matrix characteristics were similar between groups. We conclude that the AFF group had a duration-dependent increase in DMB leading to a significantly higher DMB than the non-AFF. Because BPs have high affinity to bone mineral and lining the walls of the osteocyte lacunae, the accumulation of matrix-bound BPs in AFF could lead to inhibition of the osteocyte cytoskeleton blunting their response to mechanical strains, a hypothesis to be further investigated. © 2021 American Society for Bone and Mineral Research (ASBMR).

No evidence for alteration in early secondary mineralization by either alendronate, teriparatide or combination of both in transiliac bone biopsy samples from postmenopausal osteoporotic patients

The influence of treatment with alendronate (ALN), teriparatide (TPTD) or concurrent treatment with both on the human bone matrix mineralization has not yet been fully elucidated. For this purpose we analyzed quadruple fluorochrome labelled transiliac bone biopsy samples (n = 66) from postmenopausal osteoporotic women with prior and ongoing ALN (ALN-Rx arm) or without ALN (Rx-Naïve arm) after 7 months treatment with cyclic or daily TPTD or without TPTD using quantitative backscattered electron imaging and confocal scanning laser microscopy. Additionally to the bone mineralization density distribution (BMDD) of entire cancellous and cortical compartments, we measured the mineralization kinetics, i.e. the calcium concentration between the younger (Ca_DL2) and older double labels (Ca_DL1), and in interstitial bone (Ca_int) in a subset of the biopsy cohort.We found the BMDD from the patients with prior and ongoing ALN generally shifted to higher calcium concentrations compared to those without ALN (average degree of mineralization in cancellous bone Cn.CaMean + 3.1%, p<0.001). The typical BMDD changes expected by cyclic or daily TPTD treatment due to the increased bone turnover/formation, e.g. an increase in low mineralized bone area were not observed. Additionally, we found no influence of treatment with ALN or TPTD or combination thereof on Ca_DL2, Ca_DL1, or Ca_int. Pooling the information from all groups, Ca_DL1 was +5.9% (p<0.001) higher compared to Ca_DL2, corresponding to a mineralization rate of 0.18 wt% Ca per week during the early secondary mineralization process.Our data suggest that the patients in the ALN-Rx arm had more highly mineralized bone matrix than those without ALN due to their lower bone turnover. The reason for the unexpected BMDD findings in the TPTD treated remain unknown and cannot be attributed to altered mineralization kinetics as no differences in the time course of early secondary mineralization were observed between the treatment groups.

Multimodal X-ray imaging of nanocontainer-treated macrophages and calcium distribution in the perilacunar bone matrix

Studies of biological systems typically require the application of several complementary methods able to yield statistically-relevant results at a unique level of sensitivity. Combined X-ray fluorescence and ptychography offer excellent elemental and structural imaging contrasts at the nanoscale. They enable a robust correlation of elemental distributions with respect to the cellular morphology. Here we extend the applicability of the two modalities to higher X-ray excitation energies, permitting iron mapping. Using a long-range scanning setup, we applied the method to two vital biomedical cases. We quantified the iron distributions in a population of macrophages treated with Mycobacterium-tuberculosis-targeting iron-oxide nanocontainers. Our work allowed to visualize the internalization of the nanocontainer agglomerates in the cytosol. From the iron areal mass maps, we obtained a distribution of antibiotic load per agglomerate and an average areal concentration of nanocontainers in the agglomerates. In the second application we mapped the calcium content in a human bone matrix in close proximity to osteocyte lacunae (perilacunar matrix). A concurrently acquired ptychographic image was used to remove the mass-thickness effect from the raw calcium map. The resulting ptychography-enhanced calcium distribution allowed then to observe a locally lower degree of mineralization of the perilacunar matrix.

Dnmt3a-Mediated DNA Methylation Changes Regulate Osteogenic Differentiation of hMSCs Cultivated in the 3D Scaffolds under Oxidative Stress.

Oxidative stress (OS) caused by multiple factors occurs after the implantation of bone repair materials. DNA methylation plays an important role in the regulation of osteogenic differentiation. Moreover, recent studies suggest that DNA methyltransferases (Dnmts) are involved in bone formation and resorption. However, the effect and mechanism of DNA methylation changes induced by OS on bone formation after implantation still remain unknown. Three-dimensional (3D) cell culture systems are much closer to the real situation than traditional monolayer cell culture systems in mimicking the in vivo microenvironment. We have developed porous 3D scaffolds composed of mineralized collagen type I, which mimics the composition of the extracellular matrix of human bone. Here, we first established a 3D culture model of human mesenchymal stem cells (hMSCs) seeded in the biomimetic scaffolds using 160 μM H2O2 to simulate the microenvironment of osteogenesis after implantation. Our results showed that decreased methylation levels of ALP and RUNX2 were induced by H2O2 treatment in hMSCs cultivated in the 3D scaffolds. Furthermore, we found that Dnmt3a was significantly downregulated in a porcine anterior lumbar interbody fusion model and was confirmed to be reduced by H2O2 treatment using the 3D in vitro model. The hypomethylation of ALP and RUNX2 induced by H2O2 treatment was abolished by Dnmt3a overexpression. Moreover, our findings demonstrated that the Dnmt inhibitor 5-AZA can enhance osteogenic differentiation of hMSCs under OS, evidenced by the increased expression of ALP and RUNX2 accompanied by the decreased DNA methylation of ALP and RUNX2. Taken together, these results suggest that Dnmt3a-mediated DNA methylation changes regulate osteogenic differentiation and 5-AZA can enhance osteogenic differentiation via the hypomethylation of ALP and RUNX2 under OS. The biomimetic 3D scaffolds combined with 5-AZA and antioxidants may serve as a promising novel strategy to improve osteogenesis after implantation.

Histological Criteria that Distinguish Human and Mouse Bone Formed Within a Mouse Skeletal Repair Defect.

The effectiveness of autologous cell-based skeletal repair continues to be controversial in part because in vitro predictors of in vivo human bone formation by cultured human progenitor cells are not reliable. To assist in the development of in vivo assays of human osteoprogenitor potential, a fluorescence-based histology of nondecalcified mineralized tissue is presented that provides multiple criteria to distinguish human and host osteoblasts, osteocytes, and accumulated bone matrix in a mouse calvarial defect model. These include detection of an ubiquitously expressed red fluorescent protein reporter by the implanted human cells, antibodies specific to human bone sialoprotein and a human nuclear antigen, and expression of a bone/fibroblast restricted green fluorescent protein reporter in the host tissue. Using low passage bone marrow-derived stromal cells, robust human bone matrix formation was obtained. However, a striking feature is the lack of mouse bone marrow investment and osteoclasts within the human bone matrix. This deficiency may account for the accumulation of a disorganized human bone matrix that has not undergone extensive remodeling. These features, which would not be appreciated by traditional decalcified paraffin histology, indicate the human bone matrix is not undergoing active remodeling and thus the full differentiation potential of the implanted human cells within currently used mouse models is not being realized.

Development of a demineralized and decellularized human epiphyseal bone scaffold for tissue engineering: A histological study.

Extracellular matrix (ECM) controls cellular behaviors such as proliferation, migration, and differentiation. The decellularized matrix of mammalian tissues has been used as a scaffold for the repair and reconstruction of tissue defects. In this study, for the first time demineralized and decellularized human epiphyseal bone matrix was used as a scaffold for a bone generation. Human epiphyseal bone was demineralized by hydrochloric acid and then decellularized by three methods of physical (slow freezing and snap freeze-thaw), enzymatic (trypsin 0.25%, 18 h) and chemical sodium dodecyl sulfate (SDS),)2.5%, 26 h). The scaffolds were cultured with rat adherent bone marrow cells (RABMC). Then, the histological studies were performed on days 7, 14, 21, and 28 of the culture to observe the distribution and morphology of cells. Bone formation was also investigated using Alizarin red staining. The results of this study indicated that RABMC migrated, proliferated and separated by forming lacuna in this three-dimensional bone scaffold. In addition, the Alizarin red staining indicated the calcium deposition on the scaffold in both bone differentiation and standard culture medium. The natural characteristic of the present bone scaffold, its cell adhesion features and capability to induce bone mineralization, even in the standard culture medium, provides a potentially optimal bone scaffold for bone tissue engineering.

Humanization of bone and bone marrow in an orthotopic site reveals new potential therapeutic targets in osteosarcoma

BackgroundExisting preclinical murine models often fail to predict effects of anti-cancer drugs. In order to minimize interspecies-differences between murine hosts and human bone tumors of in vivo xenograft platforms, we tissue-engineered a novel orthotopic humanized bone model. MethodsOrthotopic humanized tissue engineered bone constructs (ohTEBC) were fabricated by 3D printing of medical-grade polycaprolactone scaffolds, which were seeded with human osteoblasts and embedded within polyethylene glycol-based hydrogels containing human umbilical vein endothelial cells (HUVECs). Constructs were then implanted at the femur of NOD-scid and NSG mice. NSG mice were then bone marrow transplanted with human CD34 + cells. Human osteosarcoma (OS) growth was induced within the ohTEBCs by direct injection of Luc-SAOS-2 cells. Tissues were harvested for bone matrix and marrow morphology analysis as well as tumor biology investigations. Tumor marker expression was analyzed in the humanized OS and correlated with the expression in 68 OS patients utilizing tissue micro arrays (TMA). ResultsAfter harvesting the femurs micro computed tomography and immunohistochemical staining showed an organ, which had all features of human bone. Around the original mouse femur new bone trabeculae have formed surrounded by a bone cortex. Staining for human specific (hs) collagen type-I (hs Col-I) showed human extracellular bone matrix production. The presence of nuclei staining positive for human nuclear mitotic apparatus protein 1 (hs NuMa) proved the osteocytes residing within the bone matrix were of human origin. Flow cytometry verified the presence of human hematopoietic cells. After injection of Luc-SAOS-2 cells a primary tumor and lung metastasis developed. After euthanization histological analysis showed pathognomic features of osteoblastic OS. Furthermore, the tumor utilized the previously implanted HUVECS for angiogenesis. Tumor marker expression was similar to human patients. Moreover, the recently discovered musculoskeletal gene C12orf29 was expressed in the most common subtypes of OS patient samples. ConclusionOhTEBCs represent a suitable orthotopic microenvironment for humanized OS growth and offers a new translational direction, as the femur is the most common location of OS. The newly developed and validated preclinical model allows controlled and predictive marker studies of primary bone tumors and other bone malignancies.

Organic composite-mediated surface coating of human acellular bone matrix with strontium

Acellular bone matrix (ACBM) provides an osteoconductive scaffold for bone repair, but its osteoinductivity is poor. Strontium (Sr) improves the osteoinductivity of bone implants. In this study, we developed an organic composite-mediated strontium coating strategy for ACBM scaffolds by using the ion chelating ability of carboxymethyl cellulose (CMC) and the surface adhesion ability of dopamine (DOPA). The organic coating composite, termed the CMC-DOPA-Sr composite, was synthesized under a mild condition, and its chemical structure and strontium ion chelating ability were then determined. After surface decoration, the physicochemical properties of the strontium-coated ACBM (ACBM-Sr) scaffolds were characterized, and their biocompatibility and osteoinductivity were determined in vitro and in vivo. The results showed that the CMC-DOPA-Sr composite facilitated strontium coating on the surface of ACBM scaffolds. The ACBM-Sr scaffolds possessed a sustained strontium ion release profile, exhibited good cytocompatibility, and enhanced the osteogenic differentiation of mesenchymal stem cells in vitro. Furthermore, the ACBM-Sr scaffolds showed good histocompatibility after subcutaneous implantation in nude mice. Taken together, this study provided a simple and mild strategy to realize strontium coating for ACBM scaffolds, which resulted in good biocompatibility and improved osteoinductivity.