Effects of weaving parameters on the properties of completely biological tissue-engineered vascular grafts.

Tissue-Engineered Vascular Grafts (TEVGs) made of human textiles have been recently introduced and offer remarkable biocompatibility as well as tunable mechanical properties. The approach combines the use of Cell-Assembled extracellular Matrix (CAM) threads, produced by cultured cells in vitro, with weaving, a versatile assembly method that gives fine control over graft properties. Herein, we investigated how production parameters can modify the geometrical and mechanical properties of TEVGs to better match that of native blood vessels in order to provide long-term patency. Our goals were to decrease the mechanical strength and the luminal surface profile of our first generation of woven TEVGs, while maintaining low transmural permeability and good suture retention strength. Different TEVGs were produced by varying CAM sheet strength as well as weaving parameters such as warp count, weft ribbons width, and weft tension. An optimized design reduced the burst pressure by 35%, wall thickness by 38% and increased compliance by 269%. The improved TEVG had properties closer to that of native blood vessels, with a burst pressure of 3492 mmHg, a wall thickness of 0.69 mm, and a compliance of 4.8%/100 mmHg, while keeping excellent suture retention strength (4.7 N) and low transmural permeability (24 mL·min-1·cm-2). Moreover, the new design reduced the luminal surface profile by 48% and utilized 47% less CAM. With a comparable design, the use of decellularized CAM threads, instead of devitalized ones, led to TEVGs with much more permeable walls and higher burst pressure. The next step is to implant this optimized graft in an allogeneic sheep model of arteriovenous shunt to assess its in vivo remodeling and performance. .

Development and characterization of biological sutures made of cell-assembled extracellular matrix.

Most vascular surgical repair procedures, such as vessel anastomoses, requires using suture materials that are mechanically efficient and accepted by the patient's body. These materials are essentially composed of synthetic polymers, such as polypropylene (ProleneTM) or polyglactin (VicrylTM). However, once implanted in patients, they are recognized as foreign bodies, and the patient's immune system will degrade, encapsulate, or even expel them. In this study, we developed innovative biological sutures for cardiovascular surgical repairs using Cell-Assembled extracellular Matrix (CAM)-based ribbons. After a mechanical characterization of the CAM-based ribbons, sutures were made with hydrated or twisted/dried ribbons with an initial width of 2 or 3 mm. These biological sutures were mechanically characterized and used to anastomoseex vivoanimal aortas. Data showed that our biological sutures display lower permeability and higher burst resistance than standard ProleneTMsuture material.In vivocarotid anastomoses realized in sheep demonstrated that our biological sutures are compatible with standard vascular surgery techniques. Echography confirmed the absence of thrombus and perfect homeostasis with no blood leakage was obtained within the first 10 min after closing the anastomosis. Finally, our findings confirmed the effectiveness and clinical relevance of these innovative biological sutures.

The cell-assembled extracellular matrix: A focus on the storage stability and terminal sterilization of this human "bio" material.

The Cell-Assembled extracellular Matrix (CAM) is an attractive biomaterial because it provided the backbone of vascular grafts that were successfully implanted in patients, and because it can now be assembled in "human textiles". For future clinical development, it is important to consider key manufacturing questions. In this study, the impact of various storage conditions and sterilization methods were evaluated. After 1 year of dry frozen storage, no change in mechanical nor physicochemical properties were detected. However, storage at 4 °C and room temperature resulted in some mechanical changes, especially for dry CAM, but physicochemical changes were minor. Sterilization modified CAM mechanical and physicochemical properties marginally except for hydrated gamma treatment. All sterilized CAM supported cell proliferation. CAM ribbons were implanted subcutaneously in immunodeficient rats to assess the impact of sterilization on the innate immune response. Sterilization accelerated strength loss but no significant difference could be shown at 10 months. Very mild and transient inflammatory responses were observed. Supercritical CO2 sterilization had the least effect. In conclusion, the CAM is a promising biomaterial since it is unaffected by long-term storage in conditions available in hospitals (hydrated at 4 °C), and can be sterilized terminally (scCO2) without compromising in vitro nor in vivo performance. STATEMENT OF SIGNIFICANCE: In the field of tissue engineering, the use of extracellular matrix (ECM) proteins as a scaffolding biomaterial has become very popular. Recently, many investigators have focused on ECM produced by cells in vitro to produce unprocessed biological scaffolds. As this new kind of "biomaterial" becomes more and more relevant, it is critical to consider key manufacturing questions to facilitate future transition to the clinic. This article presents an extensive evaluation of long-term storage stability and terminal sterilization effects on an extracellular matrix assembled by cells in vitro. We believe that this article will be of great interest to help tissue engineers involved in so-called scaffold-free approaches to better prepare the translation from benchtop to bedside.

Current biofabrication methods for vascular tissue engineering and an introduction to biological textiles.

Cardiovascular diseases are the leading cause of mortality in the world and encompass several important pathologies, including atherosclerosis. In the cases of severe vessel occlusion, surgical intervention using bypass grafts may be required. Synthetic vascular grafts provide poor patency for small-diameter applications (< 6 mm) but are widely used for hemodialysis access and, with success, larger vessel repairs. In very small vessels, such as coronary arteries, synthetics outcomes are unacceptable, leading to the exclusive use of autologous (native) vessels despite their limited availability and, sometimes, quality. Consequently, there is a clear clinical need for a small-diameter vascular graft that can provide outcomes similar to native vessels. Many tissue-engineering approaches have been developed to offer native-like tissues with the appropriate mechanical and biological properties in order to overcome the limitations of synthetic and autologous grafts. This review overviews current scaffold-based and scaffold-free approaches developed to biofabricate tissue-engineered vascular grafts (TEVGs) with an introduction to the biological textile approaches. Indeed, these assembly methods show a reduced production time compared to processes that require long bioreactor-based maturation steps. Another advantage of the textile-inspired approaches is that they can provide better directional and regional control of the TEVG mechanical properties.

Towards harmonization of directly measured free 25-hydroxyvitamin D using an enzyme-linked immunosorbent assay.

The majority of circulating 25-hydroxyvitamin D (25(OH)D) is protein bound and perhaps less available than the free fraction of 25(OH)D; therefore, researchers have proposed that the measurement of free 25(OH)D in human serum may be a better indicator of vitamin D health status than total 25(OH)D. The availability of a new enzyme-linked immunosorbent assay (ELISA) for the determination of free 25(OH)D provides a method for direct measurement of the low levels of non-protein bound 25(OH)D. As an initial step towards harmonization of measurements of free 25(OH)D, the ELISA was used to measure free 25(OH)D in three existing Standard Reference Materials (SRMs): SRM 972a Vitamin D Metabolites in Frozen Human Serum, SRM 2973 Vitamin D Metabolites in Frozen Human Serum (High Level), and SRM 1949 Frozen Prenatal Human Serum. Target values for free 25(OH)D in the nine SRM serum pools, obtained by combining the results from two laboratories, ranged from 3.76 ± 0.36 to 10.0 ± 0.58 pg/mL. Of particular significance is the assignment of free 25(OH)D target values to SRM 1949, which consists of four serum pools from non-pregnant female donors of reproductive age and pregnant women in each of the three trimesters and which also has values assigned for vitamin D binding protein, which increases during pregnancy. The availability of target values for free 25(OH)D in these SRMs will allow researchers to validate new analytical methods and to compare their results with other researchers as an initial step towards harmonization of measurements among different studies and laboratories.

Development of a vascular substitute produced by weaving yarn made from human amniotic membrane.

Because synthetic vascular prostheses perform poorly in small-diameter revascularization, biological vascular substitutes are being developed as an alternative. Although theirin vivoresults are promising, their production involves long, complex, and expensive tissue engineering methods. To overcome these limitations, we propose an innovative approach that combines the human amniotic membrane (HAM), which is a widely available and cost-effective biological raw material, with a rapid and robust textile-inspired assembly strategy. Fetal membranes were collected after cesarean deliveries at term. Once isolated by dissection, HAM sheets were cut into ribbons that could be further processed by twisting into threads. Characterization of the HAM yarns (both ribbons and threads) showed that their physical and mechanical properties could be easily tuned. Since our clinical strategy will be to provide an off-the-shelf allogeneic implant, we studied the effects of decellularization and/or gamma sterilization on the histological, mechanical, and biological properties of HAM ribbons. Gamma irradiation of hydrated HAMs, with or without decellularization, did not interfere with the ability of the matrix to support endothelium formationin vitro. Finally, our HAM-based, woven tissue-engineered vascular grafts (TEVGs) exhibited clinically relevant mechanical properties. Thus, this study demonstrates that human, completely biological, allogeneic, small-diameter TEVGs can be produced from HAM, thereby avoiding costly cell culture and bioreactors.

Long-term results of autologous scaffold-free tissue-engineered vascular graft for hemodialysis access.

BACKGROUND: The growing size of the end stage renal disease (ESRD) population highlights the need for effective dialysis access. Exhausted native vascular access options have led to increased use of catheters and prosthetic shunts, which are both associated with high risks of access failure and infection. Emerging alternatives include tissue-engineered vascular grafts (TEVG). Here we present the endpoint results for 10 ESRD patients with the scaffold-free tissue-engineered vascular access produced from sheets of extracellular matrix produced in vitro by human cells in culture. METHODS: Grafts were implanted as arteriovenous shunts in 10 ESRD patients with a complex history of access failure. Follow-up included ultrasound control of graft morphology and function, dialysis efficiency, access failure, intervention rate, as well as immunohistochemical analysis of graft structure. RESULTS: One patient died of unrelated causes and three shunts failed to become useable access grafts during the 3-month maturation phase. The 12-month primary and secondary patency for the other six shunts was 86%. Survival of six shunts functioning as the vascular access was 22 ± 12 months with longest primary patency of 38.6 months. The dialysis event rate of 3.34 per patient-year decreased significantly with the use of this TEVG to 0.67. CONCLUSIONS: This living autologous tissue-engineered vascular graft seems to be an alternative to synthetic vascular access options, exhibiting advantages of native arteriovenous fistula.

Inter-donor variability of extracellular matrix production in long-term cultures of human fibroblasts.

Several tissue engineering approaches are based on the ability of mesenchymal cells to endogenously synthesize an extracellular matrix (ECM) in vitro, which can be seen as a form of biomaterial. Accordingly, the inter-donor variability of cell-assembled extracellular matrix (CAM) production is a key parameter to understand in order to progress towards clinical applications, especially for autologous strategies. In this study, CAMs were produced, under good manufacturing process conditions, from skin fibroblasts of 21 patients as part of a clinical trial to evaluate a tissue-engineered vascular graft. The inter-donor variability of CAM strength, thickness, hydroxyproline, and glycosaminoglycan was substantial (coefficient of variability of 33%, 19%, 24%, and 19%, respectively), but a significant correlation was observed between all four properties (Pearson r: 0.43 to 0.70; p-value ≤ 0.05). A CAM matrisome analysis, performed by mass spectrometry, revealed the presence of 70 ECM-related proteins. Our study shows that the relative abundance of 16 proteins (15 non-collagenous) correlated with CAM thickness. These proteins also correlated with CAM hydroxyproline content, as well as 21 other proteins that included fibrillar collagens and non-collagenous proteins. However, data demonstrated that only the relative abundance of type I collagen subunit alpha-1 was correlated to CAM strength. This study is the most extensive evaluation of CAM inter-donor variability to date and will help tissue engineers working with this type of biomaterial to design strategies that take into account this variability, especially for autologous tissue manufacturing.

Comparison of amniotic membrane versus the induced membrane for bone regeneration in long bone segmental defects using calcium phosphate cement loaded with BMP-2.

Thanks to its biological properties, the human amniotic membrane (HAM) combined with a bone substitute could be a single-step surgical alternative to the two-step Masquelet induced membrane (IM) technique for regeneration of critical bone defects. However, no study has directly compared these two membranes. We first designed a 3D-printed scaffold using calcium phosphate cement (CPC). We assessed its suitability in vitro to support human bone marrow mesenchymal stromal cells (hBMSCs) attachment and osteodifferentiation. We then performed a rat femoral critical size defect to compare the two-step IM technique with a single-step approach using the HAM. Five conditions were compared. Group 1 was left empty. Group 2 received the CPC scaffold loaded with rh-BMP2 (CPC/BMP2). Group 3 and 4 received the CPC/BMP2 scaffold covered with lyophilized or decellularized/lyophilized HAM. Group 5 underwent a two- step induced membrane procedure with insertion of a polymethylmethacrylate (PMMA) spacer followed by, after 4 weeks, its replacement with the CPC/BMP2 scaffold wrapped in the IM. Micro-CT and histomorphometric analysis were performed after six weeks. Results showed that the CPC scaffold supported the proliferation and osteodifferentiation of hBMSCs in vitro. In vivo, the CPC/BMP2 scaffold very efficiently induced bone formation and led to satisfactory healing of the femoral defect, in a single-step, without autograft or the need for any membrane covering. In this study, there was no difference between the two-step induced membrane procedure and a single step approach. However, the results indicated that none of the tested membranes further enhanced bone healing compared to the CPC/BMP2 group.

In vivo remodeling of human cell-assembled extracellular matrix yarns.

Cell-assembled extracellular matrix (CAM) has been used to produce vascular grafts. While these completely biological vascular grafts performed well in clinical trials, the in vivo remodeling and inflammatory response of this truly "bio" material has not yet been investigated. In this study, human CAM yarns were implanted subcutaneously in nude rats to investigate the innate immune response to this matrix. The impact of processing steps relevant to yarn manufacturing was evaluated (devitalization, decellularization, gamma sterilization, and twisting). We observed that yarns were still present after six months, and were integrated into a non-inflamed loose connective tissue. The CAM was repopulated by fibroblastic cells and blood vessels. While other yarns caused minor peripheral inflammation at an early stage (two weeks of implantation), gamma sterilization triggered a more intense host response dominated by the presence of M1 macrophages. The inflammatory response was resolved at six months. Yarn mechanical strength was decreased two weeks after implantation except for the more compact "twisted" yarn. While the strength of other yarns was stable after initial remodeling, the gamma-sterilized yarn continued to lose mechanical strength over time and was weaker than devitalized (control) yarns at six months. This is the first study to formally demonstrate that devitalized human CAM is very long-lived in vivo and does not trigger a degradative response, but rather is very slowly remodeled. This data supports a strategy to produce human textiles from CAM yarn for regenerative medicine applications where a scaffold with low inflammation and long-term mechanical properties are critical.

Cell-assembled extracellular matrix (CAM) sheet production: Translation from using human to large animal cells.

We have created entirely biological tissue-engineered vascular grafts (TEVGs) using sheets of cell-assembled extracellular matrix (CAM) produced by human fibroblasts in vitro. A large animal TEVG would allow long-term pre-clinical studies in a clinically relevant setting (graft size and allogeneic setting). Therefore, canine, porcine, ovine, and human skin fibroblasts were compared for their ability to form CAM sheets. Serum sourcing greatly influenced CAM production in a species-dependent manner. Ovine cells produced the most homogenous and strongest animal CAM sheets but remained ≈3-fold weaker than human sheets despite variations of serum, ascorbate, insulin, or growth factor supplementations. Key differences in cell growth dynamics, tissue development, and tissue architecture and composition were observed between human and ovine. This study demonstrates critical species-to-species differences in fibroblast behavior and how they pose a challenge when attempting to substitute animal cells for human cells during the development of tissue-engineered constructs that require long-term cultures.

Assessment of fresh and preserved amniotic membrane for guided bone regeneration in mice.

Thanks to its biological properties, the human amniotic membrane (HAM) can be used as a barrier membrane for guided bone regeneration (GBR). However, no study has assessed the influence of the preservation method of HAM for this application. This study aimed to establish the most suitable preservation method of HAM for GBR. Fresh (F), cryopreserved (C) lyophilized (L), and decellularized and lyophilized (DL) HAM were compared. The impact of preservation methods on collagen and glycosaminoglycans (GAG) content was evaluated using Masson's trichrome and alcian blue staining. Their suture retention strengths were assessed. In vitro, the osteogenic potential of human bone marrow mesenchymal stromal cells (hBMSCs) cultured on the four HAMs was evaluated using alkaline phosphatase staining and alizarin red quantification assay. In vivo, the effectiveness of fresh and preserved HAMs for GBR was assessed in a mice diaphyseal bone defect after 1 week or 1 month healing. Micro-CT and histomorphometric analysis were performed. The major structural components of HAM (collagen and GAG) were preserved whatever the preservation method used. The tearing strength of DL-HAM was significantly higher. In vitro, hBMSCs seeded on DL-HAM displayed a stronger ALP staining, and alizarin red staining quantification was significantly higher at Day 14. In vivo, L-HAM and DL-HAM significantly enhanced early bone regeneration. One month after the surgery, only DL-HAM slightly promoted bone regeneration. Several preserving methods of HAM have been studied for bone regeneration. Here, we have demonstrated that DL-HAM achieved the most promising results for GBR.

Human textiles: A cell-synthesized yarn as a truly "bio" material for tissue engineering applications.

In the field of tissue engineering, many groups have come to rely on the extracellular matrix produced by cells as the scaffold that provides structure and strength to the engineered tissue. We have previously shown that sheets of Cell-Assembled extracellular Matrix (CAM), which are entirely biological yet robust, can be mass-produced for clinical applications using normal, adult, human fibroblasts. In this article, we demonstrate that CAM yarns can be generated with a range of physical and mechanical properties. We show that this material can be used as a simple suture to close a wound or can be assembled into fully biological, human, tissue-engineered vascular grafts (TEVGs) that have high mechanical strength and are implantable. By combining this truly "bio" material with a textile-based assembly, this original tissue engineering approach is highly versatile and can produce a variety of strong human textiles that can be readily integrated in the body. STATEMENT OF SIGNIFICANCE: Yarn of synthetic biomaterials have been turned into textiles for decades because braiding, knitting and weaving machines can mass-produce medical devices with a wide range of shapes and mechanical properties. Here, we show that robust, completely biological, and human yarn can be produced by normal cells in vitro. This yarn can be used as a simple suture material or to produce the first human textiles. For example, we produced a woven tissue-engineered vascular grafts with burst pressure, suture retention strength and transmural permeability that surpassed clinical requirements. This novel strategy holds the promise of a next generation of medical textiles that will be mechanically strong without any foreign scaffolding, and will have the ability to truly integrate into the host's body.

Comparison of the impact of preservation methods on amniotic membrane properties for tissue engineering applications.

Human amniotic membrane (hAM) is considered as an attractive biological scaffold for tissue engineering. For this application, hAM has been mainly processed using cryopreservation, lyophilization and/or decellularization. However, no study has formally compared the influence of these treatments on hAM properties. The aim of this study was to develop a new decellularization-preservation process of hAM, and to compare it with other conventional treatments (fresh, cryopreserved and lyophilized). The hAM was decellularized (D-hAM) using an enzymatic method followed by a detergent decellularization method, and was then lyophilized and gamma-sterilized. Decellularization was assessed using DNA staining and quantification. D-hAM was compared to fresh (F-hAM), cryopreserved (C-hAM) and lyophilized/gamma-sterilized (L-hAM) hAM. Their cytotoxicity on human bone marrow mesenchymal stem cells (hBMSCs) and their biocompatibility in a rat subcutaneous model were also evaluated. The protocol was effective as judged by the absence of nuclei staining and the residual DNA lower than 50 ng/mg. Histological staining showed a disruption of the D-hAM architecture, and its thickness was 84% lower than fresh hAM (p < 0.001). Despite this, the labeling of type IV and type V collagen, elastin and laminin were preserved on D-hAM. Maximal force before rupture of D-hAM was 92% higher than C-hAM and L-hAM (p < 0.01), and D-hAM was 37% more stretchable than F-hAM (p < 0.05). None of the four hAM were cytotoxic, and D-hAM was the most suitable scaffold for hBMSCs proliferation. Finally, D-hAM was well integrated in vivo. In conclusion, this new hAM decellularization process appears promising for tissue engineering applications.

Cartography of the mechanical properties of the human amniotic membrane.

Because of its low immunogenicity, biological properties, and high availability, the Human Amniotic Membrane (HAM) is widely used in the clinic and in tissue engineering research. However, while its biological characteristics are well described, its mechanical properties remain understudied especially in terms of inter- and intra-HAM variability. To guide bioengineers in the use of this natural biomaterial, a detailed cartography of the HAM's mechanical properties was performed. Maximal force (Fmax) and strain at break (Smax) were identified as the relevant mechanical criteria for this study after a combined analysis of histological sections, thickness measurements after dehydration, and uniaxial tensile tests. Eight HAMs were studied by mechanical cartography using a standardized cutting protocol and sampling pattern. On average, 103 ±â€¯10 samples were retrieved and tested per HAM. Intra-tissue variability highlighted the fact that there were two mechanically distinct areas (placental and peripheral) in each HAM. For all HAMs, placental HAM was significantly stronger by 82 ±â€¯45% and more stretchable by 19 ±â€¯6% than their peripheral counterparts. Our results also demonstrated that placental, but not peripheral, HAM presented isotropic mechanical properties. Thus, placental HAM can be a raw material of choice that could be favored especially in the development of tissue engineering products where mechanical properties play a key role.

Characterization of a Cell-Assembled extracellular Matrix and the effect of the devitalization process.

We have previously shown that the Cell-Assembled extracellular Matrix (CAM) synthesized by normal, human, skin fibroblasts in vitro can be assembled in a completely biological vascular graft that was successfully tested in the clinic. The goal of this study was to perform a detailed analysis of the composition and the organization of this truly bio-material. In addition, we investigated whether the devitalization process (dehydration) used to store the CAM, and thus, make the material available "off-the-shelf," could negatively affect its organization and mechanical properties. We demonstrated that neither the thickness nor the mechanical strength of CAM sheets were significantly changed by the dehydration/freezing/rehydration cycle. The identification of over 50 extracellular matrix proteins highlighted the complex composition of the CAM. Histology showed intense collagen and glycosaminoglycan staining throughout the CAM sheet. The distribution of collagen I, collagen VI, thrombospondin-1, fibronectin-1, fibrillin-1, biglycan, decorin, lumican and versican showed various patterns that were not affected by the devitalization process. Transmission electron microscopy analysis revealed that the remarkably dense collagen network was oriented in the plane of the sheet and that neither fibril density nor diameter was changed by devitalization. Second harmonic generation microscopy revealed an intricate, multi-scale, native-like collagen fiber orientation. In conclusion, this bio-material displayed many tissue-like properties that could support normal cell-ECM interactions and allow implantation without triggering degradative responses from the host's innate immune system. This is consistent with its success in vivo. In addition, the CAM can be devitalized without affecting its mechanical or unique biological architecture. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) defines biological function and mechanical properties of tissues and organs. A number of promising tissue engineering approaches have used processed ECM from cadaver/animal tissues or cell-assembled ECM in vitro combined with scaffolds. We have shown the clinical potential of a scaffold-free approach based on an entirely biological material produced by human cells in culture without chemical processing. Here, we perform a comprehensive analysis of the properties of what can truly be called a bio-material. We also demonstrate that this material can be stored dried without losing its remarkable biological architecture.

Characterization of printed PLA scaffolds for bone tissue engineering.

Autografts remain the gold standard for orthopedic transplantations. However, to overcome its limitations, bone tissue engineering proposes new strategies. This includes the development of new biomaterials such as synthetic polymers, to serve as scaffold for tissue production. The objective of this present study was to produce poly(lactic) acid (PLA) scaffolds of different pore size using fused deposition modeling (FDM) technique and to evaluate their physicochemical and biological properties. Structural, chemical, mechanical, and biological characterizations were performed. We successfully fabricated scaffolds of three different pore sizes. However, the pore dimensions were slightly smaller than expected. We found that the 3D printing process induced decreases in both, PLA molecular weight and degradation temperatures, but did not change the semicrystalline structure of the polymer. We did not observe any effect of pore size on the mechanical properties of produced scaffolds. After the sterilization by γ irradiation, scaffolds did not exhibit any cytotoxicity towards human bone marrow stromal cells (HBMSC). Finally, after three and seven days of culture, HBMSC showed high viability and homogenous distribution irrespective of pore size. Thus, these results suggest that FDM technology is a fast and reproducible technique that can be used to fabricate tridimensional custom-made scaffolds for tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 887-894, 2018.

A Direct Assay for Measuring Free 25-Hydroxyvitamin D.

Recent studies suggest that the concentration and genotype of vitamin D binding protein (VDBP) are important factors that determine the bioavailability of 25-hydroxyvitamin D [25(OH)D] in blood. Accumulating data indicate that, e.g., in pregnant women, hemodialysis patients, chronic kidney disease, liver failure, and bladder and pancreatic cancers, the measurement of free 25(OH)D in serum provides more relevant diagnostic information than measurement of total 25(OH)D. The aim of this study was to develop and validate an ELISA for direct measurement of free 25(OH)D in serum. A simple and direct ELISA was developed, based on a two-step immunoassay procedure performed in a microtiter plate. The assay has been characterized in terms of precision (4-10% CV, according to concentration), sensitivity (limits of blank = 0.5-1.0 pg/mL and LODs = 1.3-1.8 pg/mL), accuracy (correlation to dialysis, ELISA = 0.99xdialysis-0.5 pg/mL, r2 = 0.74), cross-reactivity of the antibody for the D2 form (77%), and addition of both VDBP and albumin (35-38% recovery upon addition of VDBP, 53-58% upon addition of albumin). The assay has already been used in multiple studies, including its comparison with calculation methods and in studies of patients with liver failure, different ethnic groups, supplemented mice, respiratory diseases, and obesity. The free 25(OH)D ELISA can be used in studies as a valuable tool to establish the clinical relevance of free 25(OH)D.

[3D bioprinting in regenerative medicine and tissue engineering]. / Impression 3D en médecine régénératrice et ingénierie tissulaire.

Additive manufacturing covers a number of fashionable technologies that attract the interest of researchers in biomaterials and tissue engineering. Additive manufacturing applied to regenerative medicine covers two main areas: 3D printing and biofabrication. If 3D printing has penetrated the world of regenerative medicine, bioassembly and bioimprinting are still in their infancy. The objective of this paper is to make a non-exhaustive review of these different complementary aspects of additive manufacturing in restorative and regenerative medicine or for tissue engineering.