Bone-tissue regeneration induced by biomimetic bioactive materials is the most promising approach alternative to the clinical ones used to treat bone loss caused by trauma or diseases such as osteoporosis. The goal is to design nanostructured bioactive constructs able to reproduce the physiological environment: By mimicking the natural features of bone tissue, the cell behavior during the regeneration process may be addressed. At present, 3D-printing technologies are the only techniques able to design complex structures avoiding constraints of final shape and porosity. However, this type of biofabrication requires complex optimization of biomaterial formulations in terms of specific rheological and mechanical properties while preserving high biocompatibility. In this work, we combined nano-sized mesoporous bioactive glasses enriched with strontium ions with type I collagen, to formulate a bioactive ink for 3D-printing technologies. Moreover, to avoid the premature release of strontium ions within the crosslinking medium and to significantly increase the material mechanical and thermal stability, we applied an optimized chemical treatment using ethanol-dissolved genipin solutions. The high biocompatibility of the hybrid system was confirmed by using MG-63 and Saos-2 osteoblast-like cell lines, further highlighting the great potential of the innovative nanocomposite for the design of bone-like scaffolds.

Bone marrow aspirate (BMA) is a common source for harvesting mesenchymal stem cells (MSCs), other progenitor cells, and associated cytokines and growth factors to be used in the biologic treatment of various orthopaedic pathologies. The aspirate is commonly centrifuged into a concentrated volume that can be immediately administered to a patient using commercially available kits. However, the handling and efficacy of BMA concentrate (BMAC) are still controversial. To characterize BMA versus BMAC for MSC quantity, potency, and cytokine profile. Controlled laboratory study. From 8 participants (age, 17-68 years), 30 mL of bone marrow was aspirated by a single surgeon from either the proximal humerus or distal femur and was separated into 2 equal samples. One sample was kept as BMA, and the other half was centrifuged into BMAC. The 2 samples then underwent flow cytometry for detection of MSCs, cell analysis for colony-forming units (CFUs), and cytokine profiling. A 2-tailed t test was used to detect differences between MSCs, CFUs, and cytokine density concentrations between BMA and BMAC. The average concentration of MSCs in both BMA and BMAC was 0.001%. Average MSC events detected by flow cytometry were significantly higher in BMA versus BMAC (15.1 and 8.1, respectively; P < .045). Expanded MSCs demonstrated similar phenotypes, but CFUs were significantly increased in BMA compared with BMAC (104 vs 68 CFUs, respectively; P < .001). Total protein concentration and cytokine profiling demonstrated great variability between BMA and BMAC and between patients. Most importantly, BMAC failed to concentrate MSCs in 6 of 8 samples. There is great variability in MSC concentration, total protein concentration, and cytokine profile between BMA and BMAC. When studying the clinical efficacy of BMAC, one must also evaluate the sample itself to determine the presence, concentration, and potency of MSCs if this is to be considered a cell-based therapy. Further standard operating procedures need to be investigated to ensure reproducible results and appropriate treatments.

In bone regenerative strategies, the controlled release of growth factors is one of the main aspects for successful tissue regeneration. Recent trends in the drug delivery field increased the interest in the development of biodegradable systems able to protect and transport active agents. In the present study, we designed degradable poly(lactic-co-glycolic)acid (PLGA) nanocarriers suitable for the release of Transforming Growth Factor-beta 1 (TGF-β1), a key molecule in the management of bone cells behaviour. Spherical TGF-β1-containing PLGA (PLGA_TGF-β1) nanoparticles (ca.250 nm) exhibiting high encapsulation efficiency (ca.64%) were successfully synthesized. The TGF-β1 nanocarriers were subsequently combined with type I collagen for the fabrication of nanostructured 3D printed scaffolds able to mimic the TGF-β1 presence in the human bone extracellular matrix (ECM). The homogeneous hybrid formulation underwent a comprehensive rheological characterisation in view of 3D printing. The 3D printed collagen-based scaffolds (10 mm × 10 mm × 1 mm) successfully mimicked the TGF-β1 presence in human bone ECM as assessed by immunohistochemical TGF-β1 staining, covering ca.3.4% of the whole scaffold area. Moreover, the collagenous matrix was able to reduce the initial burst release observed in the first 24 h from about 38% for the PLGA_TGF-β1 alone to 14.5%, proving that the nanocarriers incorporation into collagen allows achieving sustained release kinetics.

Background: Mesenchymal stem cells (MSCs) are promising for cell-based therapies targeting a wide range of diseases. However, challenges in translating MSC-based therapies to clinical applications necessitate standardized protocols following Good Manufacturing Practices (GMP) guidelines. This study aimed at developing GMP-complained protocols for FPMSCs isolation and manipulation, necessary for translational research, by (1) optimize culture of MSCs derived from an infrapatellar fat pad (FPMSC) condition through animal-free media comparison and (2) establish feasibility of MSC isolation, manufacturing and storage under GMP-compliance (GMP-FPMSC). Methods: FPMSCs from three different patients were isolated following established protocols and the efficacy of two animal component-free media formulations in the culturing media were evaluated. The impact of different media formulations on cell proliferation, purity, and potency of MSCs was evaluated through doubling time, colony forming unit assay, and percentage of MSCs, respectively. Furthermore, the isolation and expansion of GMP-FPMSCs from four additional donors were optimized and characterized at each stage according to GMP requirements. Viability and sterility were checked using Trypan Blue and Bact/Alert, respectively, while purity and identity were confirmed using Endotoxin, Mycoplasma assays, and Flow Cytometry. The study also included stability assessments post-thaw and viability assessment to determine the shelf-life of the final GMP-FPMSC product. Statistical analyses were conducted using one-way ANOVA with Tukey’s Multiple Comparisons. Results: The study demonstrated that FPMSCs exhibited enhanced proliferation rates when cultured in MSC-Brew GMP Medium compared to standard MSC media. Cells cultured in this media showed lower doubling times across passages, indicating increased proliferation. Additionally, higher colony formation in FPMSCs cultured in MSC-Brew GMP Medium were observed, supporting enhanced potency. Data from our GMP validation, including cells from 4 different donors, showed post-thaw GMP-FPMSC maintained stem cell marker expression and all the specifications required for product release, including > 95% viability (> 70% is required) and sterility, even after extended storage (up to 180 days), demonstrating the reproducibility and potential of GMP-FPMSCs for clinical use as well as the robustness of the isolation and storage protocols. Conclusions: The study underscores the feasibility of FPMSCs for clinical uses under GMP conditions and emphasizes the importance of optimized culture protocols to improve cell proliferation and potency in MSC-based therapies.

Silica-based mesoporous systems have gained great interest in drug delivery applications due to their excellent biocompatibility and high loading capability. However, these materials face challenges in terms of pore-size limitations since they are characterized by nanopores ranging between 6–8 nm and thus unsuitable to host large molecular weight molecules such as proteins, enzymes and growth factors (GFs). In this work, for an application in the field of bone regeneration, large-pore mesoporous silicas (LPMSs) were developed to vehicle large biomolecules and release them under a pH stimulus. Considering bone remodeling, the proposed pH-triggered mechanism aims to mimic the release of GFs encased in the bone matrix due to bone resorption by osteoclasts (OCs) and the associated pH drop. To this aim, LPMSs were prepared by using 1,3,5-trimethyl benzene (TMB) as a swelling agent and the synthesis solution was hydrothermally treated and the influence of different process temperatures and durations on the resulting mesostructure was investigated. The synthesized particles exhibited a cage-like mesoporous structure with accessible pores of diameter up to 23 nm. LPMSs produced at 140 °C for 24 h showed the best compromise in terms of specific surface area, pores size and shape and hence, were selected for further experiments. Horseradish peroxidase (HRP) was used as model protein to evaluate the ability of the LPMSs to adsorb and release large biomolecules. After HRP-loading, LPMSs were coated with a pH-responsive polymer, poly(ethylene glycol) (PEG), allowing the release of the incorporated biomolecules in response to a pH decrease, in an attempt to mimic GFs release in bone under the acidic pH generated by the resorption activity of OCs. The reported results proved that PEG-coated carriers released HRP more quickly in an acidic environment, due to the protonation of PEG at low pH that catalyzes polymer hydrolysis reaction. Our findings indicate that LPMSs could be used as carriers to deliver large biomolecules and prove the effectiveness of PEG as pH-responsive coating. Finally, as proof of concept, a collagen-based suspension was obtained by incorporating PEG-coated LPMS carriers into a type I collagen matrix with the aim of designing a hybrid formulation for 3D-printing of bone scaffolds.

Focal chondral lesions of the knee are the most frequent type of trauma in younger patients and are associated with a high risk of developing early posttraumatic osteoarthritis. The only current clinical solutions include microfracture, osteochondral grafting, and autologous chondrocyte implantation. Cartilage tissue engineering based on biomimetic scaffolds has become an appealing strategy to repair cartilage defects. Here, a chondrogenic collagen‐chondroitin sulfate scaffold is tested in an orthotopic Lapine in vivo model to understand the beneficial effects of the immunomodulatory biomaterial on the full chondral defect. Using a combination of noninvasive imaging techniques, histological and whole transcriptome analysis, the scaffolds are shown to enhance the formation of cartilaginous tissue and suppression of host cartilage degeneration, while also supporting tissue integration and increased tissue regeneration over a 12 weeks recovery period. The results presented suggest that biomimetic materials could be a clinical solution for cartilage tissue repair, due to their ability to modulate the immune environment in favor of regenerative processes and suppression of cartilage degeneration.