Biofabrication of nanocomposite-based scaffolds containing human bone extracellular matrix for the differentiation of skeletal stem and progenitor cells.

Autograft or metal implants are routinely used in skeletal repair. However, they fail to provide long-term clinical resolution, necessitating a functional biomimetic tissue engineering alternative. The use of native human bone tissue for synthesizing a biomimetic material ink for three-dimensional (3D) bioprinting of skeletal tissue is an attractive strategy for tissue regeneration. Thus, human bone extracellular matrix (bone-ECM) offers an exciting potential for the development of an appropriate microenvironment for human bone marrow stromal cells (HBMSCs) to proliferate and differentiate along the osteogenic lineage. In this study, we engineered a novel material ink (LAB) by blending human bone-ECM (B) with nanoclay (L, Laponite®) and alginate (A) polymers using extrusion-based deposition. The inclusion of the nanofiller and polymeric material increased the rheology, printability, and drug retention properties and, critically, the preservation of HBMSCs viability upon printing. The composite of human bone-ECM-based 3D constructs containing vascular endothelial growth factor (VEGF) enhanced vascularization after implantation in an ex vivo chick chorioallantoic membrane (CAM) model. The inclusion of bone morphogenetic protein-2 (BMP-2) with the HBMSCs further enhanced vascularization and mineralization after only seven days. This study demonstrates the synergistic combination of nanoclay with biomimetic materials (alginate and bone-ECM) to support the formation of osteogenic tissue both in vitro and ex vivo and offers a promising novel 3D bioprinting approach to personalized skeletal tissue repair. Supplementary Information: The online version contains supplementary material available at 10.1007/s42242-023-00265-z.

De Novo Design of Functional Coassembling Organic-Inorganic Hydrogels for Hierarchical Mineralization and Neovascularization.

Synthetic nanostructured materials incorporating both organic and inorganic components offer a unique, powerful, and versatile class of materials for widespread applications due to the distinct, yet complementary, nature of the intrinsic properties of the different constituents. We report a supramolecular system based on synthetic nanoclay (Laponite, Lap) and peptide amphiphiles (PAs, PAH3) rationally designed to coassemble into nanostructured hydrogels with high structural integrity and a spectrum of bioactivities. Spectroscopic and scattering techniques and molecular dynamic simulation approaches were harnessed to confirm that PAH3 nanofibers electrostatically adsorbed and conformed to the surface of Lap nanodisks. Electron and atomic force microscopies also confirmed an increase in diameter and surface area of PAH3 nanofibers after coassembly with Lap. Dynamic oscillatory rheology revealed that the coassembled PAH3-Lap hydrogels displayed high stiffness and robust self-healing behavior while gas adsorption analysis confirmed a hierarchical and heterogeneous porosity. Furthermore, this distinctive structure within the three-dimensional (3D) matrix provided spatial confinement for the nucleation and hierarchical organization of high-aspect ratio hydroxyapatite nanorods into well-defined spherical clusters within the 3D matrix. Applicability of the organic-inorganic PAH3-Lap hydrogels was assessed in vitro using human bone marrow-derived stromal cells (hBMSCs) and ex vivo using a chick chorioallantoic membrane (CAM) assay. The results demonstrated that the organic-inorganic PAH3-Lap hydrogels promote human skeletal cell proliferation and, upon mineralization, integrate with the CAM, are infiltrated by blood vessels, stimulate extracellular matrix production, and facilitate extensive mineral deposition relative to the controls.

Regulation of osteoblast development by Bcl-2-associated athanogene-1 (BAG-1).

BCL-2-associated athanogene-1 (BAG-1) is expressed by osteoblast-lineage cells; early embryonic lethality in Bag-1 null mice, however, has limited the investigation of BAG-1 function in osteoblast development. In the present study, bone morphogenetic protein-2/BMP-2-directed osteogenic differentiation of bone marrow stromal cells (BMSCs) of Bag-1(+/-) (heterozygous) female mice was decreased significantly. Genes crucial for osteogenic differentiation, bone matrix formation and mineralisation were expressed at significantly lower levels in cultures of Bag-1(+/-) BMSCs supplemented with BMP-2, while genes with roles in inhibition of BMP-2-directed osteoblastogenesis were significantly upregulated. 17-ß-estradiol (E2) enhanced responsiveness of BMSCs of wild-type and Bag-1(+/-) mice to BMP-2, and promoted robust BMP-2-stimulated osteogenic differentiation of BMSCs. BAG-1 can modulate cellular responses to E2 by regulating the establishment of functional estrogen receptors (ERs), crucially, via its interaction with heat shock proteins (HSC70/HSP70). Inhibition of BAG-1 binding to HSC70 by the small-molecule chemical inhibitor, Thioflavin-S, and a short peptide derived from the C-terminal BAG domain, which mediates binding with the ATPase domain of HSC70, resulted in significant downregulation of E2/ER-facilitated BMP-2-directed osteogenic differentiation of BMSCs. These studies demonstrate for the first time the significance of BAG-1-mediated protein-protein interactions, specifically, BAG-1-regulated activation of ER by HSC70, in modulation of E2-facilitated BMP-2-directed osteoblast development.

Episomal plasmid-based generation of induced pluripotent stem cells from fetal femur-derived human mesenchymal stromal cells.

Human bone mesenchymal stromal cells derived from fetal femur 55 days post-conception were reprogrammed to induced pluripotent stem cells using episomal plasmid-based expression of OCT4, SOX2, NANOG, LIN28, SV40LT, KLF4 and c-MYC and supplemented with the following pathway inhibitors - TGFß receptor inhibitor (A-83-01), MEK inhibitor (PD325901), GSK3ß inhibitor (CHIR99021) and ROCK inhibitor (HA-100). Successful induction of pluripotency in two iPS-cell lines was demonstrated in vitro and by the Pluritest.

Site-Dependent Reference Point Microindentation Complements Clinical Measures for Improved Fracture Risk Assessment at the Human Femoral Neck.

In contrast to traditional approaches to fracture risk assessment using clinical risk factors and bone mineral density (BMD), a new technique, reference point microindentation (RPI), permits direct assessment of bone quality; in vivo tibial RPI measurements appear to discriminate patients with a fragility fracture from controls. However, it is unclear how this relates to the site of the most clinically devastating fracture, the femoral neck, and whether RPI provides information complementary to that from existing assessments. Femoral neck samples were collected at surgery after low-trauma hip fracture (n = 46; 17 male; aged 83 [interquartile range 77-87] years) and compared, using RPI (Biodent Hfc), with 16 cadaveric control samples, free from bone disease (7 male; aged 65 [IQR 61-74] years). A subset of fracture patients returned for dual-energy X-ray absorptiometry (DXA) assessment (Hologic Discovery) and, for the controls, a micro-computed tomography setup (HMX, Nikon) was used to replicate DXA scans. The indentation depth was greater in femoral neck samples from osteoporotic fracture patients than controls (p < 0.001), which persisted with adjustment for age, sex, body mass index (BMI), and height (p < 0.001) but was site-dependent, being less pronounced in the inferomedial region. RPI demonstrated good discrimination between fracture and controls using receiver-operating characteristic (ROC) analyses (area under the curve [AUC] = 0.79 to 0.89), and a model combining RPI to clinical risk factors or BMD performed better than the individual components (AUC = 0.88 to 0.99). In conclusion, RPI at the femoral neck discriminated fracture cases from controls independent of BMD and traditional risk factors but dependent on location. The clinical RPI device may, therefore, supplement risk assessment and requires testing in prospective cohorts and comparison between the clinically accessible tibia and the femoral neck. © 2015 American Society for Bone and Mineral Research.

The effects of 1α, 25-dihydroxyvitamin D3 and transforming growth factor-ß3 on bone development in an ex vivo organotypic culture system of embryonic chick femora.

Transforming growth factor-beta3 (TGF-ß3) and 1α,25-dihydroxyvitamin D3 (1α,25 (OH) 2D3) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH)2D3 and TGF-ß3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH) 2D3 (25 nM) or TGF-ß3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (µCT), histology and immunohistochemistry. 1α,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-ß3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH) 2D3 and TGF-ß3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH)2D and TGF-ß3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

Embossing of micropatterned ceramics and their cellular response.

The aim of this work is to investigate the use of microtopographies in providing physical cues to modulate the cellular response of human mesenchymal stem cells on ceramics. Two microgrooved patterns (100 µm/50 µm, 10 µm/10 µm groove/pitch) were transcribed reversely onto alumina green ceramic tapes via an embossing technique followed by sintering. Characterization of the micropatterned alumina surfaces and their cellular response was carried out. Spread and polygonal cell morphologies were observed on the wider groove (50 µm/100 µm) surface. Cells seeded onto the narrow groove (10 µm/10 µm) surface aligned themselves alongside the grooves, resulting in more elongated cell morphology. More osteoid matrix nodules shown by osteopontin and osteocalcin biomarkers were detected on the larger grooved surfaces after cell culture of 21 days, indicating a greater level of osteogenicity. This study has shown that micropatterned wider groove (50 µm) topographies are more suitable surfaces for improving osseointegration of ceramic implants.

Taking tissue engineering principles into theatre: retrieval analysis from a clinically translated case.

AIM: Tissue engineering has enormous potential for the regeneration of bone defects. Approximately 4 years ago we reported on a 62 year old patient who underwent treatment of a benign cyst in the proximal femur by impaction bone grafting supplemented with autologous bone marrow. The cyst and symptoms subsequently recurred and this patient has now required a total hip replacement. This has provided a rare opportunity for ex vivo analysis of clinically applied tissue engineered bone. MATERIALS & METHODS: The femoral head was retrieved at surgery and the structural and functional characteristics of the tissue engineered bone were analyzed by micro-computed tomography, histology and mechanical testing. RESULTS: The impacted bone demonstrated a trabecular structure that contained islands of nonincorporated graft. The graft was denser than the patient's trabecular bone with comparable strength. The cyst material had penetrated along the channel of bone and an increased number of osteoclasts were observed. DISCUSSION: This study has provided detailed ex vivo analysis of retrieved human tissue engineered bone and possible reasons for the observed construct failure are discussed in this article. The impacted bone displayed some evidence of remodeled trabecular structure, although the bone marrow aspirate that was initially combined with the allograft contained a relatively low concentration of osteoprogenitor cells. Cellular augmentation was insufficient to overcome the osteoclastic process associated with renewed cyst formation. Concentration or culture expansion of osteoprogenitor cells from aspirated bone marrow is recommended for biological augmentation of bone graft.

The epigenetic effect of glucosamine and a nuclear factor-kappa B (NF-kB) inhibitor on primary human chondrocytes--implications for osteoarthritis.

OBJECTIVE: Idiopathic osteoarthritis is the most common form of osteoarthritis (OA) world-wide and remains the leading cause of disability and the associated socio-economic burden in an increasing aging population. Traditionally, OA has been viewed as a degenerative joint disease characterized by progressive destruction of the articular cartilage and changes in the subchondral bone culminating in joint failure. However, the etiology of OA is multifactorial involving genetic, mechanical and environmental factors. Treatment modalities include analgesia, joint injection with steroids or hyaluronic acid, oral supplements including glucosamine and chondroitin sulfate, as well as physiotherapy. Thus, there is significant interest in the discovery of disease modifying agents. One such agent, glucosamine (GlcN) is commonly prescribed even though the therapeutic efficacy and mechanism of action remain controversial. Inflammatory cytokines, including IL-1ß, and proteinases such as MMP-13 have been implicated in the pathogenesis and progression of OA together with an associated CpG demethylation in their promoters. We have investigated the potential of GlcN to modulate NF-kB activity and cytokine-induced abnormal gene expression in articular chondrocytes and, critically, whether this is associated with an epigenetic process. METHOD: Human chondrocytes were isolated from the articular cartilage of femoral heads, obtained with ethical permission, following fractured neck of femur surgery. Chondrocytes were cultured for 5 weeks in six separate groups; (i) control culture, (ii) cultured with a mixture of 2.5 ng/ml IL-1ß and 2.5 ng/ml oncostatin M (OSM), (iii) cultured with 2mM N-acetyl GlcN (Sigma-Aldrich), (iv) cultured with a mixture of 2.5 ng/ml IL-1ß, 2.5 ng/ml OSM and 2mM GlcN, (v) cultured with 1.0 µM BAY 11-7082 (BAY; NF-kB inhibitor: Calbiochem, Darmstadt, Germany) and, (vi) cultured with a mixture of 2.5 ng/ml IL-1ß, 2.5 ng/ml OSM and 1.0 µM BAY. The levels of IL1B and MMP13 mRNA were examined using qRT-PCR. The percentage DNA methylation in the CpG sites of the IL1ß and MMP13 proximal promoter were quantified by pyrosequencing. RESULT: IL1ß expression was enhanced over 580-fold in articular chondrocytes treated with IL-1ß and OSM. GlcN dramatically ameliorated the cytokine-induced expression by 4-fold. BAY alone increased IL1ß expression by 3-fold. In the presence of BAY, IL-1ß induced IL1B mRNA levels were decreased by 6-fold. The observed average percentage methylation of the -256 CpG site in the IL1ß promoter was 65% in control cultures and decreased to 36% in the presence of IL-1ß/OSM. GlcN and BAY alone had a negligible effect on the methylation status of the IL1B promoter. The cytokine-induced loss of methylation status in the IL1B promoter was ameliorated by both GlcN and BAY to 44% and 53%, respectively. IL-1ß/OSM treatment increased MMP13 mRNA levels independently of either GlcN or BAY and no change in the methylation status of the MMP13 promoter was observed. CONCLUSION: We demonstrate for the first time that GlcN and BAY can prevent cytokine-induced demethylation of a specific CpG site in the IL1ß promoter and this was associated with decreased expression of IL1ß. These studies provide a potential mechanism of action for OA disease modifying agents via NF-kB and, critically, demonstrate the need for further studies to elucidate the role that NF-kB may play in DNA demethylation in human chondrocytes.

Fabrication of pillar-like titania nanostructures on titanium and their interactions with human skeletal stem cells.

Surface nanotopography is known to influence the interaction of human skeletal (mesenchymal) stem cells (hMSC) with a material surface. While most surface nanopatterning has been performed on polymer-based surfaces there is a need for techniques to produce well-defined topography features with tuneable sizes on relevant load-bearing implant materials such as titanium (Ti). In this study titania nanopillar structures with heights of either 15, 55 or 100 nm were produced on Ti surfaces using anodization through a porous alumina mask. The influence of the surface structure heights on hMSC adhesion, spreading, cytoskeletal formation and differentiation was examined. The 15 nm high topography features resulted in the greatest cell response with bone matrix nodule forming on the Ti surface after 21 days.

The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder.

A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.