Olive Mill Wastewater as Source of Polyphenols with Nutraceutical Properties.

BACKGROUND: Agrifood waste products are often considered rich sources of bioactive compounds that can be conveniently recovered. Due to these peculiar characteristics, the study of these waste products is attracting great interest in nutraceutical research. Olive mill wastewaters (OMWWs) are generated by extra virgin olive oil (EVOO) production, and they pose environmental challenges due to their disposal. This study aimed to characterize the polyphenolic profile and to evaluate the nutraceutical properties of OMWW extracts from two Tuscan olive cultivars, Leccino (CL) and Frantoio (CF), collected during different time points in EVOO production. METHOD: After a liquid-liquid extraction, the HPLC and LC-MS/MS analysis of OMWW extracts confirmed the presence of 18 polyphenolic compounds. RESULTS: The polyphenol composition varied between the cultivars and during maturation stages. Notably, oleacein was detected at remarkably high levels in CL1 and CF1 extracts (314.628 ± 19.535 and 227.273 ± 3.974 µg/mg, respectively). All samples demonstrated scavenging effects on free radicals (DPPH and ABTS assays) and an anti-inflammatory potential by inhibiting cyclooxygenase (COX) enzymes. CONCLUSIONS: This study highlights the nutraceutical potential of OMWW extracts, emphasizing their antioxidant, antiradical, and anti-inflammatory activities. The results demonstrate the influence of olive cultivar, maturation stage, and extraction process on the polyphenolic composition and the bioactivity of OMWW extracts. These findings support a more profitable reuse of OMWW as an innovative, renewable, and low-cost source of dietary polyphenols with potential applications as functional ingredients in the development of dietary supplements, as well as in the pharmaceutical and cosmetics industries.

Assessment of Electrospun Poly(ε-caprolactone) and Poly(lactic acid) Fiber Scaffolds to Generate 3D In Vitro Models of Colorectal Adenocarcinoma: A Preliminary Study.

Three-dimensional scaffold-based culture has been increasingly gaining influence in oncology as a therapeutic strategy for tumors with a high relapse percentage. This study aims to evaluate electrospun poly(ε-caprolactone) (PCL) and poly(lactic acid) (PLA) scaffolds to create a 3D model of colorectal adenocarcinoma. Specifically, the physico-mechanical and morphological properties of PCL and PLA electrospun fiber meshes collected at different drum velocities, i.e., 500 rpm, 1000 rpm and 2500 rpm, were assessed. Fiber size, mesh porosity, pore size distribution, water contact angle and tensile mechanical properties were investigated. Caco-2 cells were cultured on the produced PCL and PLA scaffolds for 7 days, demonstrating good cell viability and metabolic activity in all the scaffolds. A cross-analysis of the cell-scaffold interactions with morphological, mechanical and surface characterizations of the different electrospun fiber meshes was carried out, showing an opposite trend of cell metabolic activity in PLA and PCL scaffolds regardless of the fiber alignment, which increased in PLA and decreased in PCL. The best samples for Caco-2 cell culture were PCL500 (randomly oriented fibers) and PLA2500 (aligned fibers). Caco-2 cells had the highest metabolic activity in these scaffolds, with Young's moduli in the range of 8.6-21.9 MPa. PCL500 showed Young's modulus and strain at break close to those of the large intestine. Advancements in 3D in vitro models of colorectal adenocarcinoma could move forward the development of therapies for this cancer.

Cell-free biomimetic polyurethane-based scaffold for breast reconstruction following non-malignant lesion resection. A first-in-human study.

BACKGROUND: Based on the volume of tissue removed, conservative surgery (BCS) cannot always guarantee satisfactory cosmetic results, unless resorting to more complex oncoplastic approaches. Investigating an alternative to optimize aesthetic outcomes minimizing surgical complexity, was the purpose of this study. We assessed an innovative surgical procedure based on the use of a biomimetic polyurethane-based scaffold intended for regenerating soft-tissue resembling fat, in patients undergoing BCS for non-malignant breast lesions. Safety and performance of the scaffold, and safety and feasibility of the entire implant procedure were evaluated. METHODS: A volunteer sample of 15 female patients underwent lumpectomy with immediate device positioning, performing seven study visits with six-month follow-up. We evaluated incidence of adverse events (AEs), changes in breast appearance (using photographs and anthropomorphic measurements), interference with ultrasound and MRI (assessed by two independent investigators), investigator's satisfaction (through a VAS scale), patient's pain (through a VAS scale) and quality of life (QoL) (using the BREAST-Q© questionnaire). Data reported are the results of the interim analysis on the first 5 patients. RESULTS: No AEs were device related nor serious. Breast appearance was unaltered and the device did not interference with imaging. High investigator's satisfaction, minimal post-operative pain and positive impact on QoL were also detected. CONCLUSIONS: Albeit on a limited number of patients, data showed positive outcomes both in terms of safety and performance, paving the way to an innovative breast reconstructive approach with a potential remarkable impact on clinical application of tissue engineering. TRIAL REGISTRATION: ClinicalTrials.gov (NCT04131972, October 18, 2019).

Biomaterial-Assisted 3D In Vitro Tumor Models: From Organoid towards Cancer Tissue Engineering Approaches.

Cancers are a leading cause of death around the world, accounting for nearly 10 million deaths yearly [...].

Electrospun Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate)/Olive Leaf Extract Fiber Mesh as Prospective Bio-Based Scaffold for Wound Healing.

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters synthesized by various microorganisms. Due to their biocompatibility and biodegradation, PHAs have been proposed for biomedical applications, including tissue engineering scaffolds. Olive leaf extract (OLE) can be obtained from agri-food biowaste and is a source of polyphenols with remarkable antioxidant properties. This study aimed at incorporating OLE inside poly(hydroxybutyrate-co-hydroxyvalerate) (PHBHV) fibers via electrospinning to obtain bioactive bio-based blends that are useful in wound healing. PHBHV/OLE electrospun fibers with a size of 1.29 ± 0.34 µm were obtained. Fourier transform infrared chemical analysis showed a uniform surface distribution of hydrophilic -OH groups, confirming the presence of OLE in the electrospun fibers. The main OLE phenols were released from the fibers within 6 days. The biodegradation of the scaffolds in phosphate buffered saline was investigated, demonstrating an adequate stability in the presence of metalloproteinase 9 (MMP-9), an enzyme produced in chronic wounds. The scaffolds were preliminarily tested in vitro with HFFF2 fibroblasts and HaCaT keratinocytes, suggesting adequate cytocompatibility. PHBHV/OLE fiber meshes hold promising features for wound healing, including the treatment of ulcers, due to the long period of durability in an inflamed tissue environment and adequate cytocompatibility.

Combined Application of Patient-Derived Cells and Biomaterials as 3D In Vitro Tumor Models.

Although advances have been made in cancer therapy, cancer remains the second leading cause of death in the U.S. and Europe, and thus efforts to continue to study and discover better treatment methods are ongoing. Three-dimensional (3D) tumor models have shown advantages over bi-dimensional (2D) cultures in evaluating the efficacy of chemotherapy. This commentary aims to highlight the potential of combined application of biomaterials with patient-derived cancer cells as a 3D in vitro model for the study and treatment of cancer patients. Five studies were discussed which demonstrate and provided early evidence to create 3D models with accurate microenvironments that are comparable to in vivo tumors. To date, the use of patient-derived cells for a more personalized approach to healthcare in combination with biomaterials to create a 3D tumor is still relatively new and uncommon for application in clinics. Although highly promising, it is important to acknowledge the current limitations and challenges of developing these innovative in vitro models, including the need for biologists and laboratory technicians to become familiar with biomaterial scaffolds, and the effort for bioengineers to create easy-to-handle scaffolds for routine assessment.

An Osteosarcoma Model by 3D Printed Polyurethane Scaffold and In Vitro Generated Bone Extracellular Matrix.

Osteosarcoma is a primary bone tumor characterized by a dismal prognosis, especially in the case of recurrent disease or metastases. Therefore, tools to understand in-depth osteosarcoma progression and ultimately develop new therapeutics are urgently required. 3D in vitro models can provide an optimal option, as they are highly reproducible, yet sufficiently complex, thus reliable alternatives to 2D in vitro and in vivo models. Here, we describe 3D in vitro osteosarcoma models prepared by printing polyurethane (PU) by fused deposition modeling, further enriched with human mesenchymal stromal cell (hMSC)-secreted biomolecules. We printed scaffolds with different morphologies by changing their design (i.e., the distance between printed filaments and printed patterns) to obtain different pore geometry, size, and distribution. The printed PU scaffolds were stable during in vitro cultures, showed adequate porosity (55-67%) and tunable mechanical properties (Young's modulus ranging in 0.5-4.0 MPa), and resulted in cytocompatible. We developed the in vitro model by seeding SAOS-2 cells on the optimal PU scaffold (i.e., 0.7 mm inter-filament distance, 60° pattern), by testing different pre-conditioning factors: none, undifferentiated hMSC-secreted, and osteo-differentiated hMSC-secreted extracellular matrix (ECM), which were obtained by cell lysis before SAOS-2 seeding. Scaffolds pre-cultured with osteo-differentiated hMSCs, subsequently lysed, and seeded with SAOS-2 cells showed optimal colonization, thus disclosing a suitable biomimetic microenvironment for osteosarcoma cells, which can be useful both in tumor biology study and, possibly, treatment.

Neuron Compatibility and Antioxidant Activity of Barium Titanate and Lithium Niobate Nanoparticles.

The biocompatibility and the antioxidant activity of barium titanate (BaTiO3) and lithium niobate (LiNbO3) were investigated on a neuronal cell line, the PC12, to explore the possibility of using piezoelectric nanoparticles in the treatment of inner ear diseases, avoiding damage to neurons, the most delicate and sensitive human cells. The cytocompatibility of the compounds was verified by analysing cell viability, cell morphology, apoptotic markers, oxidative stress and neurite outgrowth. The results showed that BaTiO3 and LiNbO3 nanoparticles do not affect the viability, morphological features, cytochrome c distribution and production of reactive oxygen species (ROS) by PC12 cells, and stimulate neurite branching. These data suggest the biocompatibility of BaTiO3 and LiNbO3 nanoparticles, and that they could be suitable candidates to improve the efficiency of new implantable hearing devices without damaging the neuronal cells.

Piezoelectric Signals in Vascularized Bone Regeneration.

The demand for bone substitutes is increasing in Western countries. Bone graft substitutes aim to provide reconstructive surgeons with off-the-shelf alternatives to the natural bone taken from humans or animal species. Under the tissue engineering paradigm, biomaterial scaffolds can be designed by incorporating bone stem cells to decrease the disadvantages of traditional tissue grafts. However, the effective clinical application of tissue-engineered bone is limited by insufficient neovascularization. As bone is a highly vascularized tissue, new strategies to promote both osteogenesis and vasculogenesis within the scaffolds need to be considered for a successful regeneration. It has been demonstrated that bone and blood vases are piezoelectric, namely, electric signals are locally produced upon mechanical stimulation of these tissues. The specific effects of electric charge generation on different cells are not fully understood, but a substantial amount of evidence has suggested their functional and physiological roles. This review summarizes the special contribution of piezoelectricity as a stimulatory signal for bone and vascular tissue regeneration, including osteogenesis, angiogenesis, vascular repair, and tissue engineering, by considering different stem cell sources entailed with osteogenic and angiogenic potential, aimed at collecting the key findings that may enable the development of successful vascularized bone replacements useful in orthopedic and otologic surgery.

Combined Antimicrobial Effect of Bio-Waste Olive Leaf Extract and Remote Cold Atmospheric Plasma Effluent.

A novel strategy involving Olive Leaf Extract (OLE) and Cold Atmospheric Plasma (CAP) was developed as a green antimicrobial treatment. Specifically, we reported a preliminary investigation on the combined use of OLE + CAP against three pathogens, chosen to represent medical and food industries (i.e., E. coli, S. aureus and L. innocua). The results indicated that a concentration of 100 mg/mL (total polyphenols) in OLE can exert an antimicrobial activity, but still insufficient for a total bacterial inactivation. By using plain OLE, we significantly reduced the growth of Gram positive S. aureus and L. innocua, but not Gram-negative E. coli. Instead, we demonstrated a remarkable decontamination effect of OLE + CAP in E. coli, S. aureus and L. innocua samples after 6 h. This effect was optimally maintained up to 24 h in S. aureus strain. E. coli and L. innocua grew again in 24 h. In the latter strain, OLE alone was most effective to significantly reduce bacterial growth. By further adjusting the parameters of OLE + CAP technology, e.g., OLE amount and CAP exposure, it could be possible to prolong the initial powerful decontamination over a longer time. Since OLE derives from a bio-waste and CAP is a non-thermal technology based on ionized air, we propose OLE + CAP as a potential green platform for bacterial decontamination. As a combination, OLE and CAP can lead to better antimicrobial activity than individually and may replace or complement conventional thermal procedures in food and biomedical industries.

Waste Autochthonous Tuscan Olive Leaves (Olea europaea var. Olivastra seggianese) as Antioxidant Source for Biomedicine.

Olive leaf extract (OLE) can be obtained as biowaste and is extensively used a food supplement and an over-the-counter drug for its beneficial effects. New studies have investigated OLE concerning the role of oxidative stress in the pathogenesis of vascular disease. This in vitro study aims to evaluate if OLE extracted from the Tuscan Olea europaea protects endothelial cells against oxidative stress generated by reactive oxygen species (ROS). METHODS: OLE total polyphenols (TPs) were characterized by the Folin-Ciocalteu method. Endothelial cells were grown in conventional cultures (i.e., two-dimensional, 2D) and on a biomaterial scaffold (i.e., three-dimensional, 3D) fabricated via electrospinning. Cell viability and ROS measurement after H2O2 insults were performed. RESULTS: OLE TP content was 23.29 mg GAE/g, and oleuropein was the principal compound. The dose-dependent viability curve highlighted the absence of significant cytotoxic effects at OLE concentrations below 250 µg/mL TPs. By using OLE preconditioning at 100 µg/mL, cell viability decrease was observed, being in 3D lower than in the 2D model. OLE was protective against ROS in both models. CONCLUSIONS: OLE represents a high-value antioxidant source obtained by biowaste that is interesting for biomedical products. Using a 3D scaffold could be the best predictive model to mimic the physiological conditions of vascular tissue reaction.

Chitin Nanofibrils and Nanolignin as Functional Agents in Skin Regeneration.

Chitin and lignin, by-products of fishery and plant biomass, can be converted to innovative high value bio- and eco-compatible materials. On the nanoscale, high antibacterial, anti-inflammatory, cicatrizing and anti-aging activity is obtained by controlling their crystalline structure and purity. Moreover, electropositive chitin nanofibrlis (CN) can be combined with electronegative nanolignin (NL) leading to microcapsule-like systems suitable for entrapping both hydrophilic and lipophilic molecules. The aim of this study was to provide morphological, physico-chemical, thermogravimetric and biological characterization of CN, NL, and CN-NL complexes, which were also loaded with glycyrrhetinic acid (GA) as a model of a bioactive molecule. CN-NL and CN-NL/GA were thermally stable up to 114 °C and 127 °C, respectively. The compounds were administered to in vitro cultures of human keratinocytes (HaCaT cells) and human mesenchymal stromal cells (hMSCs) for potential use in skin contact applications. Cell viability, cytokine expression and effects on hMSC multipotency were studied. For each component, CN, NL, CN-NL and CN-NL/GA, non-toxic concentrations towards HaCaT cells were identified. In the keratinocyte model, the proinflammatory cytokines IL-1α, IL-1 ß, IL-6, IL-8 and TNF-α that resulted were downregulated, whereas the antimicrobial peptide human ß defensin-2 was upregulated by CN-LN. The hMSCs were viable, and the use of these complexes did not modify the osteo-differentiation capability of these cells. The obtained findings demonstrate that these biocomponents are cytocompatible, show anti-inflammatory activity and may serve for the delivery of biomolecules for skin care and regeneration.

3D Models of Pancreatic Ductal Adenocarcinoma via Tissue Engineering.

Cancer tissue engineering is an emerging multidisciplinary field aimed at growing cancerous cells onto porous biomaterial scaffolds and proper stimuli to ultimately reproduce 3D tumor tissue-like constructs in vitro. Unlike conventional 2D cell cultures and spheroids, these tissue models can reproduce cancer lesions very similar to those present in native tumor, and can be viable for some weeks, making it possible to study cancer biology phenomena and new therapies in a more reliable fashion than with conventional in vitro platforms. This chapter shows the preparation of a 3D model of pancreatic ductal adenocarcinoma (PDAC), including fabrication of a suitable scaffold, culture of PDAC cells on the scaffold, viability test, and histologic assessment.

Liver Cancer: Current and Future Trends Using Biomaterials.

Hepatocellular carcinoma (HCC) is the fifth most common type of cancer diagnosed and the second leading cause of death worldwide. Despite advancement in current treatments for HCC, the prognosis for this cancer is still unfavorable. This comprehensive review article focuses on all the current technology that applies biomaterials to treat and study liver cancer, thus showing the versatility of biomaterials to be used as smart tools in this complex pathologic scenario. Specifically, after introducing the liver anatomy and pathology by focusing on the available treatments for HCC, this review summarizes the current biomaterial-based approaches for systemic delivery and implantable tools for locally administrating bioactive factors and provides a comprehensive discussion of the specific therapies and targeting agents to efficiently deliver those factors. This review also highlights the novel application of biomaterials to study HCC, which includes hydrogels and scaffolds to tissue engineer 3D in vitro models representative of the tumor environment. Such models will serve to better understand the tumor biology and investigate new therapies for HCC. Special focus is given to innovative approaches, e.g., combined delivery therapies, and to alternative approaches-e.g., cell capture-as promising future trends in the application of biomaterials to treat HCC.

Raman spectroscopy of osteosarcoma cells.

Osteosarcoma is the most common primary malignant bone tumor. In the last years, several studies have demonstrated that the increase of Hydroxyapatite (HA) and Interleukin-6 (IL-6) syntheses compared to those expressed by normal osteoblasts could be used to detect the degree of malignancy of osteosarcoma cells. Conventional biochemical methods widely employed to evaluate bone cell differentiation, including normal and cancerous phenotypes, are time consuming and may require a large amount of cells. HA is a mineral form of calcium phosphate whose presence increases with maturation of osteosarcoma cells. Analogously, IL-6 is a fundamental cytokine whose production is highly increased in osteosarcoma cells. In this study, we employ Raman spectroscopy to the identification and discrimination of osteosarcoma cells from osteo-differentiated mesenchymal stromal cells (MSCs) by detecting the presence of HA and IL-6. However, while the identification of HA is facilitated by the characteristic peak at 960 cm-1, corresponding to symmetric stretching (P-O) mode, the quantification of IL-6 it is much more elusive, being its Raman signal characterized by cysteine, but also by phenylalanine, amide I II and III whose signals are common to other proteins. Supported by an accurate multivariate analysis, the results show that Raman spectroscopy is a high sensitivity technique dealing out a direct and quantitative measurement of specific mineralization levels of osteosarcoma cells. In turn, by exploiting the Surface-Enhanced Raman Scattering stimulated by internalized Gold Nanoshells (AuNSs) and combined with scanning probe microscopies, we were able to employ Raman spectroscopy to study subcellular components locally.

3D fiber deposited polymeric scaffolds for external auditory canal wall.

The external auditory canal (EAC) is an osseocartilaginous structure extending from the auricle to the eardrum, which can be affected by congenital, inflammatory, and neoplastic diseases, thus reconstructive materials are needed. Current biomaterial-based approaches for the surgical reconstruction of EAC posterior wall still suffer from resorption (biological) and extrusion (synthetic). In this study, 3D fiber deposited scaffolds based on poly(ethylene oxide terephthalate)/poly(butylene terephthalate) were designed and fabricated to replace the EAC wall. Fiber diameter and scaffold porosity were optimized, leading to 200 ± 33 µm and 55% ± 5%, respectively. The mechanical properties were evaluated, resulting in a Young's modulus of 25.1 ± 7.0 MPa. Finally, the EAC scaffolds were tested in vitro with osteo-differentiated human mesenchymal stromal cells (hMSCs) with different seeding methods to produce homogeneously colonized replacements of interest for otologic surgery. This study demonstrated the fabrication feasibility of EAC wall scaffolds aimed to match several important requirements for biomaterial application to the ear under the Tissue Engineering paradigm, including shape, porosity, surface area, mechanical properties and favorable in vitro interaction with osteoinduced hMSCs. This study demonstrated the fabrication feasibility of outer ear canal wall scaffolds via additive manufacturing. Aimed to match several important requirements for biomaterial application to ear replacements under the Tissue Engineering paradigm, including shape, porosity and pore size, surface area, mechanical properties and favorable in vitro interaction with osteo-differentiated mesenchymal stromal cells.

Ossicular replacement prostheses from banked bone with ergonomic and functional geometry.

This study shows the fabrication of innovative ossicular replacement prostheses (ORPs) based on banked cortical bone using computer numerically controlled ultraprecision micromilling, as a substantial improvement of "second generation" ORPs. Our aim is to combine optimal middle ear compatibility and surgical manageability in a single device, by releasing off-the-shelf homograft ORPs provided with the appealing features of synthetic ORPs, such as lightness, safety, measurement accuracy, surface decoration, and geometric plasticity. The new total ORP prototype was 13.1 ± 0.1 mg, leading to 81% weight reduction with respect to the previous model. Surface motifs of the head plate were applied to prevent slipping and migration after surgery, as shown by finite element modeling analysis. In addition, bone ORPs were provided with holed head plates to facilitate their surgical positioning while reducing their mass. A comparative measurement of acoustic responses of bone against synthetic partial ORPs in the 250-8000 Hz frequency range demonstrated their superior behavior. This study showed that banked compact bone can be optimally manufactured, eventually enabling the fabrication of light, standardized, and highly performant ORPs. The new bone ORPs may represent the ideal combination of biocompatibility and technology which can ultimately accomplish unmet otosurgical needs. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2495-2506, 2017.

Biomaterial-Based Implantable Devices for Cancer Therapy.

This review article focuses on the current local therapies mediated by implanted macroscaled biomaterials available or proposed for fighting cancer and also highlights the upcoming research in this field. Several authoritative review articles have collected and discussed the state-of-the-art as well as the advancements in using biomaterial-based micro- and nano-particle systems for drug delivery in cancer therapy. On the other hand, implantable biomaterial devices are emerging as highly versatile therapeutic platforms, which deserve an increased attention by the healthcare scientific community, as they are able to offer innovative, more effective and creative strategies against tumors. This review summarizes the current approaches which exploit biomaterial-based devices as implantable tools for locally administrating drugs and describes their specific medical applications, which mainly target resected brain tumors or brain metastases for the inaccessibility of conventional chemotherapies. Moreover, a special focus in this review is given to innovative approaches, such as combined delivery therapies, as well as to alternative approaches, such as scaffolds for gene therapy, cancer immunotherapy and metastatic cell capture, the later as promising future trends in implantable biomaterials for cancer applications.

Detection and localization of gold nanoshells inside cells: near-field approximation.

The optical properties of metal nanoparticles play a fundamental role for their use in a wide range of applications. In hyperthermia treatment, for example, gold nanoshells (NSs, dielectric core+gold shell) pre-embedded in a cancer cell absorb energy when exposed to appropriate wavelengths of a laser beam and heat up, thereby destroying the cancer cell. In this process, nevertheless, healthy tissues (not targeted by the NSs) along the laser path are not affected; this is because most biological soft tissues have a relatively low light absorption coefficient in the near-infrared (NIR) regions-a characteristic known as the tissue optical window. Over such a window, NIR light transmits through the tissues with scattering-limited attenuation and minimal heating, thereby avoiding damage to healthy tissues. As a consequence, the identification of NSs assumed a fundamental role for the further development of such cancer treatment. Recently, we have demonstrated the possibility to identify 100-150 nm diameter gold NSs inside mouse cells using a scanning near-optical microscope (SNOM). In this paper, we provide a numerical demonstration that the SNOM is able to locate NSs inside the cell with a particle-aperture distance of about 100 nm. This result was obtained by developing an analytical approach based on the calculation of the dyadic Green function in the near-field approximation. The implications of our findings will remarkably affect further investigations on the interaction between NSs and biological systems.

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products. Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions - namely, patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor's own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.