An investigation of composition, morphology, mechanical properties, and microdamage accumulation of human type 2 diabetic bone.

This study investigates the biomechanics of type 2 diabetic bone fragility through a multiscale experimental strategy that considers structural, mechanical, and compositional components of ex vivo human trabecular and cortical bone. Human tissue samples were obtained from the femoral heads of patients undergoing total hip replacement. Mechanical testing was carried out on isolated trabecular cores using monotonic and cyclic compression loading and nanoindentation experiments, with bone microdamage analysed using micro-computed tomography (CT) imaging. Bone composition was evaluated using Raman spectroscopy, high-performance liquid chromatography, and fluorometric spectroscopy. It was found that human type 2 diabetic bone had altered mechanical, compositional, and morphological properties compared to non-type 2 diabetic bone. High-resolution micro-CT imaging showed that cores taken from the central trabecular region of the femoral head had higher bone mineral density (BMD), bone volume, trabecular thickness, and reduced trabecular separation. Type 2 diabetic bone also had enhanced macro-mechanical compressive properties under mechanical loading compared to non-diabetic controls, with significantly higher apparent modulus, yield stress, and pre-yield toughness evident, even when properties were normalised against the bone volume. Using nanoindentation, there were no significant differences in the tissue-level mechanical properties of cortical or trabecular bone in type 2 diabetic samples compared to controls. Through compositional analysis, higher levels of furosine were found in type 2 diabetic trabecular bone, and an increase in both furosine and carboxymethyl-lysine (an advanced glycation end-product) was found in cortical bone. Raman spectroscopy showed that type 2 diabetic bone had a higher mineral-to-matrix ratio, carbonate substitution, and reduced crystallinity compared to the controls. Together, this study shows that type 2 diabetes leads to distinct changes in both organic and mineral phases of the bone tissue matrix, but these changes did not coincide with any reduction in the micro- or macro-mechanical properties of the tissue under monotonic or cyclic loading.

Expert Revision of Key Elements for Clinical-Grade Production and Qualification of Perinatal Derivatives.

Perinatal derivatives have been proposed as adjunct therapeutic strategies or innovative treatments. Undoubtedly, perinatal derivatives can offer the opportunity and source material to isolate multipotent stem cells, but both maternal- and fetal-derived tissues can be processed and transformed into engineered tissues or advanced biomedical devices, whose potential remains to be fully elucidated. Promising preclinical and clinical results collected so far clearly foresee an escalation of such novel treatments. Market forecasts predict exponential growth in such advanced medicinal products during the next decade, with a pragmatic innovation for medicine into a more advanced biomedical version, enlarging the portfolio for treating a wide range of congenital and acute conditions. However, all these promising and fascinating therapeutic possibilities cannot gain a solid and recognized role in established medical practice without rigid and harmonized manufacturing strategies. The implementation of strategies according to guidelines and directives compiled by Regulatory Agencies, in conformity to (European) Pharmacopoeia and for Good Manufacturing Practice -conforming production of such products, represent critical steps required to translate perinatal technologies into effective therapeutic approaches. During the past 5 years, a panel of European experts and developers, gathered under the umbrella of the COST Sprint Action, supported by the European Cooperation in Science and Technology action, had the opportunity to revise and summarize experience and recommendations for a fruitful and proficient generation of perinatal biomedical products. In order to facilitate the creation and potential commercialization of perinatal bioengineered and advanced pharmaceutical products and technologies, such a collection of data and recommendations is described and discussed here.

Expert Consideration on Regulatory Aspects for Perinatal Derivatives in Clinical Settings.

Perinatal derivatives (PnD) are drawing growing interest among the scientific community as an unrestricted source of multipotent stem cells, secretome, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options, but they require the development of regenerative approaches. With this development, the question of regulation of donation, processing, and distribution has therefore become more important. Within the European Cooperation in Science and Technology (COST) community, we compiled a group of international experts on PnD technologies, who revised and compared existing EU national regulations. Notably, despite clear European directives, each EU Country has developed their own implementation and standard levels for cell- and tissue-based therapies. To enable extended applications of PnD treatments within the EU community and worldwide, harmonization is highly recommended. This paper aims to provide an overview of the various options available to introduce PnD into clinical practice. For this purpose, the different aspects resulting from (1) the type of PnD, (2) the amount of available data, (3) the degree of manipulation, and (4) the intended application and the process toward a possible commercialization will be presented. In the future, it will be important to find a balance between regulatory requirements and the best medical quality of the PnD product.

Hybrid Mineral/Organic Material Induces Bone Bridging and Bone Volume Augmentation in Rat Calvarial Critical Size Defects.

In craniofacial bone defects, the promotion of bone volume augmentation remains a challenge. Finding strategies for bone regeneration such as combining resorbable minerals with organic polymers would contribute to solving the bone volume roadblock. Here, dicalcium phosphate dihydrate, chitosan and hyaluronic acid were used to functionalize a bone-side collagen membrane. Despite an increase in the release of inflammatory mediators by human circulating monocytes, the in vivo implantation of the functionalized membrane allowed the repair of a critical-sized defect in a calvaria rat model with de novo bone exhibiting physiological matrix composition and structural organization. Microtomography, histological and Raman analysis combined with nanoindentation testing revealed an increase in bone volume in the presence of the functionalized membrane and the formation of woven bone after eight weeks of implantation; these data showed the potential of dicalcium phosphate dihydrate, chitosan and hyaluronic acid to induce an efficient repair of critical-sized bone defects and establish the importance of thorough multi-scale characterization in assessing biomaterial outcomes in animal models.

Mechanical stimuli and matrix properties modulate cancer spheroid growth in three-dimensional gelatin culture.

Most patients who succumb to cancer have metastases to bone that contribute to their death. Cancer cells that metastasize to bone are regularly subjected to mechanical stimuli that may affect their proliferation, growth and protein expression. Understanding why some cancer cells thrive in this environment could provide insight into new approaches to prevent or treat metastasis to bone. We used 4T1 cells as a model of breast cancer cells, and implanted them in gelatin hydrogels with moduli of 1 or 2.7 kPa to mimic the properties of bone marrow. The constructs were subjected to either perfusion of media through the hydrogel or combined perfusion and cyclic mechanical compression for 1 h d-1 for 4 d. Controls were cultured in free-swelling conditions. The cells formed spheroids during the 4 d of culture, with larger spheroids in the statically cultured constructs than in perfusion or compressed constructs. In stiffer gelatin, smaller spheroids formed in compressed constructs than perfusion alone, while compression had no effect compared to perfusion in the softer gelatin. Immunostaining indicated that the spheroids expressed osteopontin, parathyroid hormone-related protein and fibronectin, which are all hallmarks of bone metastasis. The proliferative marker Ki67 was present in all spheroids on day 4. In the 1 kPa gelatin, Ki67 staining intensity was greater in the statically cultured, free-swelling constructs than in bioreactor culture, regardless of dynamic compression. By contrast, proliferation was higher in the compressed gelatins compared to perfusion alone in the 2.7 kPa constructs, although the spheroids were smaller, on average. This suggests the stiffer gelatin may restrict spheroid growth at the same time that it enhances mechanobiological signalling during compression. Taken together, 4T1 breast cancer cells are mechanically sensitive, and mechanical stimuli can alter their proliferation and protein expression within soft materials with mechanical properties similar to bone marrow. As such, both in vivo and in vitro models of cancer metastasis should consider the role of the mechanical environment in the bone.

ROCK-II inhibition suppresses impaired mechanobiological responses in early estrogen deficient osteoblasts.

Mechanobiological responses by osteoblasts are governed by downstream Rho-ROCK signalling through actin cytoskeleton re-arrangements but whether these responses are influenced by estrogen deficiency during osteoporosis remains unknown. The objective of this study was to determine alterations in the mechanobiological responses of estrogen-deficient osteoblasts and investigate whether an inhibitor of the Rho-ROCK signalling can revert these changes. MC3T3-E1 cells were pre-treated with 10 nM 17-ß estradiol for 7 days and further cultured with or without estradiol for next 2 days. These cells were treated with or without ROCK-II inhibitor, Y-27632, and oscillatory fluid flow (OFF, 1Pa, 0.5 Hz, 1 h) was applied. Here, we report that Prostaglandin E2 release, Runt-related transcription factor 2 and Osteopontin gene expression were significantly enhanced in response to OFF in estrogen-deficient cells than in cells with estrogen (3.73 vs 1.63 pg/ng DNA; 13.5 vs 2.6 fold, 2.1 vs 0.4 fold respectively). Upon ROCK-II inhibition, these enhanced effects of estrogen deficiency were downregulated. OFF increased the fibril anisotropy in cells pre-treated with estrogen and this increase was suppressed upon ROCK-II inhibition. This study is the first to demonstrate altered mechanobiological responses by osteoblasts during early estrogen deficiency and that these responses to OFF can be suppressed upon ROCK inhibition.

Primary Human Osteoblasts Cultured in a 3D Microenvironment Create a Unique Representative Model of Their Differentiation Into Osteocytes.

Microengineered systems provide an in vitro strategy to explore the variability of individual patient response to tissue engineering products, since they prefer the use of primary cell sources representing the phenotype variability. Traditional in vitro systems already showed that primary human osteoblasts embedded in a 3D fibrous collagen matrix differentiate into osteocytes under specific conditions. Here, we hypothesized that translating this environment to the organ-on-a-chip scale creates a minimal functional unit to recapitulate osteoblast maturation toward osteocytes and matrix mineralization. Primary human osteoblasts were seeded in a type I collagen hydrogel, to establish the role of lower (2.5 × 105 cells/ml) and higher (1 × 106 cells/ml) cell density on their differentiation into osteocytes. A custom semi-automatic image analysis software was used to extract quantitative data on cellular morphology from brightfield images. The results are showing that cells cultured at a high density increase dendrite length over time, stop proliferating, exhibit dendritic morphology, upregulate alkaline phosphatase (ALP) activity, and express the osteocyte marker dental matrix protein 1 (DMP1). On the contrary, cells cultured at lower density proliferate over time, do not upregulate ALP and express the osteoblast marker bone sialoprotein 2 (BSP2) at all timepoints. Our work reveals that microengineered systems create unique conditions to capture the major aspects of osteoblast differentiation into osteocytes with a limited number of cells. We propose that the microengineered approach is a functional strategy to create a patient-specific bone tissue model and investigate the individual osteogenic potential of the patient bone cells.

Alterations in osteocyte mediated osteoclastogenesis during estrogen deficiency and under ROCK-II inhibition: An in vitro study using a novel postmenopausal multicellular niche model.

This study sought to derive an enhanced understanding of the complex intracellular interactions that drive bone loss in postmenopausal osteoporosis. We applied an in-vitro multicellular niche to recapitulate cell-cell signalling between osteocytes, osteoblasts and osteoclasts to investigate (1) how estrogen-deficient and mechanically loaded osteocytes regulate osteoclastogenesis and (2) whether ROCK-II inhibition affects these mechanobiological responses. We report that mechanically stimulated and estrogen-deficient osteocytes upregulated RANKL/OPG and M-CSF gene expression, when compared to those treated with 10 nM estradiol. Osteoclast precursors (RAW 264.7) cultured within this niche underwent significant reduction in osteoclastogenic gene expression (CTSK), and there was an increasing trend in the area covered by TRAP+ osteoclasts (24% vs. 19.4%, p = 0.06). Most interestingly, upon treatment with the ROCK-II inhibitor, RANKL/OPG and M-CSF gene expression by estrogen-deficient osteocytes were downregulated. Yet, this inhibition of the pro-osteoclastogenic factors by osteocytes did not ultimately reduce the differentiation of osteoclast precursors. Indeed, TRAP and CTSK gene expressions in osteoclast precursors were upregulated, and there was an increased trend for osteoclast area (30.4% vs. 24%, p = 0.07), which may have been influenced by static osteoblasts (MC3T3-E1) that were included in the niche. We conclude that ROCK-II inhibition can attenuate bone loss driven by osteocytes during estrogen deficiency.

Mechanical stimulations on human bone marrow mesenchymal stem cells enhance cells differentiation in a three-dimensional layered scaffold.

Scaffolds laden with stem cells are a promising approach for articular cartilage repair. Investigations have shown that implantation of artificial matrices, growth factors or chondrocytes can stimulate cartilage formation, but no existing strategies apply mechanical stimulation on stratified scaffolds to mimic the cartilage environment. The purpose of this study was to adapt a spraying method for stratified cartilage engineering and to stimulate the biosubstitute. Human mesenchymal stem cells from bone marrow were seeded in an alginate (Alg)/hyaluronic acid (HA) or Alg/hydroxyapatite (Hap) gel to direct cartilage and hypertrophic cartilage/subchondral bone differentiation, respectively, in different layers within a single scaffold. Homogeneous or composite stratified scaffolds were cultured for 28 days and cell viability and differentiation were assessed. The heterogeneous scaffold was stimulated daily. The mechanical behaviour of the stratified scaffolds were investigated by plane-strain compression tests. Results showed that the spraying process did not affect cell viability. Moreover, cell differentiation driven by the microenvironment was increased with loading: in the layer with Alg/HA, a specific extracellular matrix of cartilage, composed of glycosaminoglycans and type II collagen was observed, and in the Alg/Hap layer more collagen X was detected. Hap seemed to drive cells to a hypertrophic chondrocytic phenotype and increased mechanical resistance of the scaffold. In conclusion, mechanical stimulations will allow for the production of a stratified biosubstitute, laden with human mesenchymal stem cells from bone marrow, which is capable in vivo to mimic all depths of chondral defects, thanks to an efficient combination of stem cells, biomaterial compositions and mechanical loading.

* Mimicking the Biochemical and Mechanical Extracellular Environment of the Endochondral Ossification Process to Enhance the In Vitro Mineralization Potential of Human Mesenchymal Stem Cells.

Chondrogenesis and mechanical stimulation of the cartilage template are essential for bone formation through the endochondral ossification process in vivo. Recent studies have demonstrated that in vitro regeneration strategies that mimic these aspects separately, either chondrogenesis or mechanical stimulation, can promote mineralization to a certain extent both in vitro and in vivo. However, to date no study has sought to incorporate both the formation of the cartilage template and the application of mechanical stimulation simultaneously to induce osteogenesis. In this study, we test the hypothesis that mimicking both the biochemical and mechanical extracellular environment arising during endochondral ossification can enhance the in vitro mineralization potential of human mesenchymal stem cells (hMSCs). hMSC aggregates were cultured for 21 days under the following culture conditions; (1) Growth Medium - hydrostatic pressure (HP), (2) Chondrogenic Priming-HP, (3) Growth Medium + HP, and (4) Chondrogenic Priming +HP. Each group was then further cultured for another 21 days in the presence of osteogenic growth factors without HP. Biochemical (DNA, sulfate glycosaminoglycan, hydroxyproline, alkaline phosphatase activity, and calcium), histological (Alcian Blue and Alizarin Red), and immunohistological (Col I, II, and X, and BSP-2) analyses were conducted to investigate chondrogenic and osteogenic differentiation at various time points (14, 21, 35, and 42 days). Our results showed the application of HP-induced chondrogenesis similar to that of chondrogenic priming, but interestingly, there was a reduction in hypertrophy markers (collagen type X) by applying HP alone versus chondrogenic priming alone. Moreover, the results showed that both chondrogenic priming and HP in tandem during the priming period, followed by culture in osteogenic medium, accelerated the osteogenic potential of hMSCs.

Active implant combining human stem cell microtissues and growth factors for bone-regenerative nanomedicine.

AIMS: Mesenchymal stem cells (MSCs) from adult bone marrow provide an exciting and promising stem cell population for the repair of bone in skeletal diseases. Here, we describe a new generation of collagen nanofiber implant functionalized with growth factor BMP-7 nanoreservoirs and equipped with human MSC microtissues (MTs) for regenerative nanomedicine. MATERIALS & METHODS: By using a 3D nanofibrous collagen membrane and by adding MTs rather than single cells, we optimize the microenvironment for cell colonization, differentiation and growth. RESULTS & CONCLUSION: Furthermore, in this study, we have shown that by combining BMP-7 with these MSC MTs in this double 3D environment, we further accelerate bone growth in vivo. The strategy described here should enhance the efficiency of therapeutic implants compared with current simplistic approaches used in the clinic today based on collagen implants soaked in bone morphogenic proteins.

A living thick nanofibrous implant bifunctionalized with active growth factor and stem cells for bone regeneration.

New-generation implants focus on robust, durable, and rapid tissue regeneration to shorten recovery times and decrease risks of postoperative complications for patients. Herein, we describe a new-generation thick nanofibrous implant functionalized with active containers of growth factors and stem cells for regenerative nanomedicine. A thick electrospun poly(ε-caprolactone) nanofibrous implant (from 700 µm to 1 cm thick) was functionalized with chitosan and bone morphogenetic protein BMP-7 as growth factor using layer-by-layer technology, producing fish scale-like chitosan/BMP-7 nanoreservoirs. This extracellular matrix-mimicking scaffold enabled in vitro colonization and bone regeneration by human primary osteoblasts, as shown by expression of osteocalcin, osteopontin, and bone sialoprotein (BSPII), 21 days after seeding. In vivo implantation in mouse calvaria defects showed significantly more newly mineralized extracellular matrix in the functionalized implant compared to a bare scaffold after 30 days' implantation, as shown by histological scanning electron microscopy/energy dispersive X-ray microscopy study and calcein injection. We have as well bifunctionalized our BMP-7 therapeutic implant by adding human mesenchymal stem cells (hMSCs). The activity of this BMP-7-functionalized implant was again further enhanced by the addition of hMSCs to the implant (living materials), in vivo, as demonstrated by the analysis of new bone formation and calcification after 30 days' implantation in mice with calvaria defects. Therefore, implants functionalized with BMP-7 nanocontainers associated with hMSCs can act as an accelerator of in vivo bone mineralization and regeneration.

Chondrogenic induction of mesenchymal stromal/stem cells from Wharton's jelly embedded in alginate hydrogel and without added growth factor: an alternative stem cell source for cartilage tissue engineering.

BACKGROUND: Due to their intrinsic properties, stem cells are promising tools for new developments in tissue engineering and particularly for cartilage tissue regeneration. Although mesenchymal stromal/stem cells from bone marrow (BM-MSC) have long been the most used stem cell source in cartilage tissue engineering, they have certain limits. Thanks to their properties such as low immunogenicity and particularly chondrogenic differentiation potential, mesenchymal stromal/stem cells from Wharton's jelly (WJ-MSC) promise to be an interesting source of MSC for cartilage tissue engineering. METHODS: In this study, we propose to evaluate chondrogenic potential of WJ-MSC embedded in alginate/hyaluronic acid hydrogel over 28 days. Hydrogels were constructed by the original spraying method. Our main objective was to evaluate chondrogenic differentiation of WJ-MSC on three-dimensional scaffolds, without adding growth factors, at transcript and protein levels. We compared the results to those obtained from standard BM-MSC. RESULTS: After 3 days of culture, WJ-MSC seemed to be adapted to their new three-dimensional environment without any detectable damage. From day 14 and up to 28 days, the proportion of WJ-MSC CD73(+), CD90(+), CD105(+) and CD166(+) decreased significantly compared to monolayer marker expression. Moreover, WJ-MSC and BM-MSC showed different phenotype profiles. After 28 days of scaffold culture, our results showed strong upregulation of cartilage-specific transcript expression. WJ-MSC exhibited greater type II collagen synthesis than BM-MSC at both transcript and protein levels. Furthermore, our work highlighted a relevant result showing that WJ-MSC expressed Runx2 and type X collagen at lower levels than BM-MSC. CONCLUSIONS: Once seeded in the hydrogel scaffold, WJ-MSC and BM-MSC have different profiles of chondrogenic differentiation at both the phenotypic level and matrix synthesis. After 4 weeks, WJ-MSC, embedded in a three-dimensional environment, were able to adapt to their environment and express specific cartilage-related genes and matrix proteins. Today, WJ-MSC represent a real alternative source of stem cells for cartilage tissue engineering.

Collagen implants equipped with 'fish scale'-like nanoreservoirs of growth factors for bone regeneration.

Implants triggering rapid, robust and durable tissue regeneration are needed to shorten recovery times and decrease risks of postoperative complications for patients. Here, we describe active living collagen implants with highly promising bone regenerative properties. Bioactivity of the implants is obtained through the protective and stabilizing layer-by-layer immobilization of a protein growth factor in association with a polysaccharide (chitosan), within the form of nanocontainers decorating the collagen nanofibers. All components of the implants are US FDA approved. From both in vitro and in vivo evaluations, the sophisticated strategy described here should enhance, at a reduced cost, the safety and efficacy of the therapeutic implants in terms of large bone defects repair compared with current simplistic approaches based on the soaking of the implants with protein growth factor.