Titanium-Doped Diamond-like Carbon Layers as a Promising Coating for Joint Replacements Supporting Osteogenic Differentiation of Mesenchymal Stem Cells.

Diamond-like carbon (DLC) layers are known for their high corrosion and wear resistance, low friction, and high biocompatibility. However, it is often necessary to dope DLC layers with additional chemical elements to strengthen their adhesion to the substrate. Ti-DLC layers (doped with 0.4, 2.1, 3.7, 6.6, and 12.8 at.% of Ti) were prepared by dual pulsed laser deposition, and pure DLC, glass, and polystyrene (PS) were used as controls. In vitro cell-material interactions were investigated with an emphasis on cell adhesion, proliferation, and osteogenic differentiation. We observed slightly increasing roughness and contact angle and decreasing surface free energy on Ti-DLC layers with increasing Ti content. Three-week biological experiments were performed using adipose tissue-derived stem cells (ADSCs) and bone marrow mesenchymal stem cells (bmMSCs) in vitro. The cell proliferation activity was similar or slightly higher on the Ti-doped materials than on glass and PS. Osteogenic cell differentiation on all materials was proved by collagen and osteocalcin production, ALP activity, and Ca deposition. The bmMSCs exhibited greater initial proliferation potential and an earlier onset of osteogenic differentiation than the ADSCs. The ADSCs showed a slightly higher formation of focal adhesions, higher metabolic activity, and Ca deposition with increasing Ti content.

Poly(octamethylene citrate) Modified with Glutathione as a Promising Material for Vascular Tissue Engineering.

One of the major goals of vascular tissue engineering is to develop much-needed materials that are suitable for use in small-diameter vascular grafts. Poly(1,8-octamethylene citrate) can be considered for manufacturing small blood vessel substitutes, as recent studies have demonstrated that this material is cytocompatible with adipose tissue-derived stem cells (ASCs) and favors their adhesion and viability. The work presented here is focused on modifying this polymer with glutathione (GSH) in order to provide it with antioxidant properties, which are believed to reduce oxidative stress in blood vessels. Cross-linked poly(1,8-octamethylene citrate) (cPOC) was therefore prepared by polycondensation of citric acid and 1,8-octanediol at a 2:3 molar ratio of the reagents, followed by in-bulk modification with 0.4, 0.8, 4 or 8 wt.% of GSH and curing at 80 °C for 10 days. The chemical structure of the obtained samples was examined by FTIR-ATR spectroscopy, which confirmed the presence of GSH in the modified cPOC. The addition of GSH increased the water drop contact angle of the material surface and lowered the surface free energy values. The cytocompatibility of the modified cPOC was evaluated in direct contact with vascular smooth-muscle cells (VSMCs) and ASCs. The cell number, the cell spreading area and the cell aspect ratio were measured. The antioxidant potential of GSH-modified cPOC was measured by a free radical scavenging assay. The results of our investigation indicate the potential of cPOC modified with 0.4 and 0.8 wt.% of GSH to produce small-diameter blood vessels, as the material was found to: (i) have antioxidant properties, (ii) support VSMC and ASC viability and growth and (iii) provide an environment suitable for the initiation of cell differentiation.

Adipose-Derived Stem Cells in Reinforced Collagen Gel: A Comparison between Two Approaches to Differentiation towards Smooth Muscle Cells.

Scaffolds made of degradable polymers, such as collagen, polyesters or polysaccharides, are promising matrices for fabrication of bioartificial vascular grafts or patches. In this study, collagen isolated from porcine skin was processed into a gel, reinforced with collagen particles and with incorporated adipose tissue-derived stem cells (ASCs). The cell-material constructs were then incubated in a DMEM medium with 2% of FS (DMEM_part), with added polyvinylalcohol nanofibers (PVA_part sample), and for ASCs differentiation towards smooth muscle cells (SMCs), the medium was supplemented either with human platelet lysate released from PVA nanofibers (PVA_PL_part) or with TGF-ß1 + BMP-4 (TGF + BMP_part). The constructs were further endothelialised with human umbilical vein endothelial cells (ECs). The immunofluorescence staining of alpha-actin and calponin, and von Willebrand factor, was performed. The proteins involved in cell differentiation, the extracellular matrix (ECM) proteins, and ECM remodelling proteins were evaluated by mass spectrometry on day 12 of culture. Mechanical properties of the gels with ASCs were measured via an unconfined compression test on day 5. Gels evinced limited planar shrinkage, but it was higher in endothelialised TGF + BMP_part gel. Both PVA_PL_part samples and TGF + BMP_part samples supported ASC growth and differentiation towards SMCs, but only PVA_PL_part supported homogeneous endothelialisation. Young modulus of elasticity increased in all samples compared to day 0, and PVA_PL_part gel evinced a slightly higher ratio of elastic energy. The results suggest that PVA_PL_part collagen construct has the highest potential to remodel into a functional vascular wall.

Biomimetic biphasic curdlan-based scaffold for osteochondral tissue engineering applications - Characterization and preliminary evaluation of mesenchymal stem cell response in vitro.

Osteochondral defects remain a huge problem in medicine today. Biomimetic bi- or multi-phasic scaffolds constitute a very promising alternative to osteochondral autografts and allografts. In this study, a new curdlan-based scaffold was designed for osteochondral tissue engineering applications. To achieve biomimetic properties, it was enriched with a protein component - whey protein isolate as well as a ceramic ingredient - hydroxyapatite granules. The scaffold was fabricated via a simple and cost-efficient method, which represents a significant advantage. Importantly, this technique allowed generation of a scaffold with two distinct, but integrated phases. Scanning electron microcopy and optical profilometry observations demonstrated that phases of biomaterial possessed different structural properties. The top layer of the biomaterial (mimicking the cartilage) was smoother than the bottom one (mimicking the subchondral bone), which is beneficial from a biological point of view because unlike bone, cartilage is a smooth tissue. Moreover, mechanical testing showed that the top layer of the biomaterial had mechanical properties close to those of natural cartilage. Although the mechanical properties of the bottom layer of scaffold were lower than those of the subchondral bone, it was still higher than in many analogous systems. Most importantly, cell culture experiments indicated that the biomaterial possessed high cytocompatibility towards adipose tissue-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells in vitro. Both phases of the scaffold enhanced cell adhesion, proliferation, and chondrogenic differentiation of stem cells (revealing its chondroinductive properties in vitro) as well as osteogenic differentiation of these cells (revealing its osteoinductive properties in vitro). Given all features of the novel curdlan-based scaffold, it is worth noting that it may be considered as promising candidate for osteochondral tissue engineering applications.

Advancements in structure-property correlation studies of cross-linked citric acid-based elastomers from the perspective of medical application.

Herein, a renewed prominence towards the synthesis of poly(alkylene citrate) (PAC) biomaterials and their detailed chemical, structural and mechanical characterization has been reported. Based on the modifications to the PAC synthesis protocol introduced in this study, the fabrication process was significantly streamlined, the reaction yields were increased, and the homogeneity of the final materials was found to be substantially improved. Comprehensive nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) studies of the fabricated prepolymers shed light on the mechanism of the PAC cross-linking process and supported the design of materials with enhanced biocompatibility. Therefore, the initial molar ratio of the reagents involved in the synthesis of PAC materials was found to be pivotal to both the biological and mechanical properties of the final products. Moreover, cell viability and proliferation assays revealed enhanced biocompatibility of the materials formulated with a molar ratio of diol over citric acid (3 : 2 mol/mol) in comparison to the most commonly described 1 : 1 analogue without affecting the possibility of further functionalization. Furthermore, this work creates a new paradigm for prospective studies on the properties of modified PAC materials and their application in medicine and tissue engineering.

Interaction between Galectin-3 and Integrins Mediates Cell-Matrix Adhesion in Endothelial Cells and Mesenchymal Stem Cells.

Galectin-3 (Gal-3) is a ß-galactoside-binding protein that influences various cell functions, including cell adhesion. We focused on the role of Gal-3 as an extracellular ligand mediating cell-matrix adhesion. We used human adipose tissue-derived stem cells and human umbilical vein endothelial cells that are promising for vascular tissue engineering. We found that these cells naturally contained Gal-3 on their surface and inside the cells. Moreover, they were able to associate with exogenous Gal-3 added to the culture medium. This association was reduced with a ß-galactoside LacdiNAc (GalNAcß1,4GlcNAc), a selective ligand of Gal-3, which binds to the carbohydrate recognition domain (CRD) in the Gal-3 molecule. This ligand was also able to detach Gal-3 newly associated with cells but not Gal-3 naturally present on cells. In addition, Gal-3 preadsorbed on plastic surfaces acted as an adhesion ligand for both cell types, and the cell adhesion was resistant to blocking with LacdiNAc. This result suggests that the adhesion was mediated by a binding site different from the CRD. The blocking of integrin adhesion receptors on cells with specific antibodies revealed that the cell adhesion to the preadsorbed Gal-3 was mediated, at least partially, by ß1 and αV integrins-namely α5ß1, αVß3, and αVß1 integrins.

The Influence of Low- and High-Negative-Pressure Liposuction and Different Harvesting Sites on the Viability and Yield of Adipocytes and Other Nucleated Cells.

BACKGROUND: The volume effect of fat grafting is highly dependent on the presence of viable adipocytes and other nucleated cells within the lipoaspirate. We suspected that one of the crucial factors influencing cell viability is the negative pressure applied during the fat graft harvesting and the suitability of various harvest sites when compared to others. Despite much discussion, there is no consensus on the optimal negative pressure or the best site for harvesting so we designed an experiment to test this. METHODS: Fat graft taken under low negative pressure (- 200 mmHg) or high negative pressure (- 700 mmHg) from the thigh or abdominal regions from 21 healthy human donors was evaluated. The principal variables studied were: a) total number and viability of nucleated cells, b) liposuction duration and c) blood admixture. Other variables studied were body mass index, the impact of age and enzymatic digestion. RESULTS: The absolute number and viability of nucleated cells and the blood admixture did not differ significantly between lipoaspirates obtained under different vacuum conditions or from different regions. The time taken to acquire the same volume of lipoaspirate was significantly increased using low negative pressure. The time taken to collect cells in the thigh region significantly increased with increasing BMI but this correlation was not found when harvesting in the abdominal region. The BMI and age did not impact the results in any of the measured variables. The enzymatic digestion rate was independent of the negative pressure used to harvest. CONCLUSION: Our results indicate that neither the negative pressure used nor the area chosen has any significant influence on the viability and yield of harvested cells. The time taken to obtain lipoaspirate using low pressure is significantly longer than when using high pressure. No significant difference was found in the value of blood admixture using different vacuum pressures, and no correlation exists between the body mass index and the cell viability or age of the patients and the time of liposuction. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine Ratings, please refer to Table of Contents or online Instructions to Authors www.springer.com/00266 .

Resistance of Winter Spelt Wheat [Triticum aestivum subsp. spelta (L.) Thell.] to Fusarium Head Blight.

Resistance to Fusarium head blight (FHB) of spelt wheat was investigated in field trials carried out at three European locations between 2016 and 2018. Resistance was assessed after artificial inoculation by visual scoring of symptoms and the determination of the contamination of grains and glumes with the mycotoxin deoxynivalenol (DON). It was found that typical spelt traits such as tall plant height, lax spikes, and tough glumes play a role as passive resistance factors. Across all test environments, modern spelt varieties with a significantly reduced plant height showed a significantly higher susceptibility to FHB and a higher contamination of the grains with DON compared to old landraces/varieties and plant genetic resources. Similarly, the lowest mycotoxin levels in grains were found only in old landraces and varieties, while the highest DON concentration was observed mainly in modern varieties. The results obtained can be used for the selection of suitable parental material for breeding spelt with improved FHB resistance.

The Influence of High and Low Negative Pressure Liposuction and Various Harvesting Techniques on the Viability and Function of Harvested Cells-a Systematic Review of Animal and Human Studies.

BACKGROUND: An understanding of fat grafting methodology, techniques and patient-related factors is crucial when considering fat grafting. Multiple factors can influence the success of a fat graft and consequently the outcome of the procedure. The aim of this systematic review is to elucidate the influence of negative pressure and various techniques of fat harvesting on the viability and function of cells, particularly adipocytes and adipose-derived stem cells. METHODS: We conducted a literature search from 1975 to 2020 using the PubMed bibliography, ScienceDirect, SCOPUS and the Google Scholar databases which produced 168,628 articles on the first pass. After applying all the exclusion criteria by two independent reviewers, we were left with 21 articles (level IV of Oxford Centre for Evidence-Based Studies and Grade C of Grade Practice Recommendation from the American Society of Plastic Surgeons) on which this review is based. RESULTS: From 11 studies focused on different negative pressures, no one found using high negative pressure advantageous. Summarising 13 studies focused on various harvesting techniques (excision, syringe, and pump-machine), most often equal results were reported, followed by excision being better than either syringe or liposuction. CONCLUSION: From our systematic review, we can conclude that the low negative pressure seems to yield better results and that the excision seems to be the most sparing method for fat graft harvesting. However, we have to point out that this conclusion is based on a very limited number of statistically challengeable articles and we recommend well-conducted further research. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Growth Factors VEGF-A165 and FGF-2 as Multifunctional Biomolecules Governing Cell Adhesion and Proliferation.

Vascular endothelial growth factor-A165 (VEGF-A165) and fibroblast growth factor-2 (FGF-2) are currently used for the functionalization of biomaterials designed for tissue engineering. We have developed a new simple method for heterologous expression and purification of VEGF-A165 and FGF-2 in the yeast expression system of Pichia pastoris. The biological activity of the growth factors was assessed in cultures of human and porcine adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). When added into the culture medium, VEGF-A165 stimulated proliferation only in HUVECs, while FGF-2 stimulated the proliferation of both cell types. A similar effect was achieved when the growth factors were pre-adsorbed to polystyrene wells. The effect of our recombinant growth factors was slightly lower than that of commercially available factors, which was attributed to the presence of some impurities. The stimulatory effect of the VEGF-A165 on cell adhesion was rather weak, especially in ADSCs. FGF-2 was a potent stimulator of the adhesion of ADSCs but had no to negative effect on the adhesion of HUVECs. In sum, FGF-2 and VEGF-A165 have diverse effects on the behavior of different cell types, which maybe utilized in tissue engineering.

Accelerated in vitro recellularization of decellularized porcine pericardium for cardiovascular grafts.

An ideal decellularized allogenic or xenogeneic cardiovascular graft should be capable of preventing thrombus formation after implantation. The antithrombogenicity of the graft is ensured by a confluent endothelial cell layer formed on its surface. Later repopulation and remodeling of the scaffold by the patient's cells should result in the formation of living autologous tissue. In the work presented here, decellularized porcine pericardium scaffolds were modified by growing a fibrin mesh on the surface and inside the scaffolds, and by attaching heparin and human vascular endothelial growth factor (VEGF) to this mesh. Then the scaffolds were seeded with human adipose tissue-derived stem cells (ASCs). While the ASCs grew only on the surface of the decellularized pericardium, the fibrin-modified scaffolds were entirely repopulated in 28 d, and the scaffolds modified with fibrin, heparin and VEGF were already repopulated within 6 d. Label free mass spectrometry revealed fibronectin, collagens, and other extracellular matrix proteins produced by ASCs during recellularization. Thin layers of human umbilical endothelial cells were formed within 4 d after the cells were seeded on the surfaces of the scaffold, which had previously been seeded with ASCs. The results indicate that an artificial tissue prepared by in vitro recellularization and remodeling of decellularized non-autologous pericardium with autologous ASCs seems to be a promising candidate for cardiovascular grafts capable of accelerating in situ endothelialization. ASCs resemble the valve interstitial cells present in heart valves. An advantage of this approach is that ASCs can easily be collected from the patient by liposuction.

Differentiation of adipose tissue-derived stem cells towards vascular smooth muscle cells on modified poly(L-lactide) foils.

The aim of our research was to study the behaviour of adipose tissue-derived stem cells (ADSCs) and vascular smooth muscle cells (VSMCs) on variously modified poly(L-lactide) (PLLA) foils, namely on pristine PLLA, plasma-treated PLLA, PLLA grafted with polyethylene glycol (PEG), PLLA grafted with dextran (Dex), and the tissue culture polystyrene (PS) control. On these materials, the ADSCs were biochemically differentiated towards VSMCs by a medium supplemented with TGFß1, BMP4 and ascorbic acid (i.e. differentiation medium). ADSCs cultured in a non-differentiation medium were used as a negative control. Mature VSMCs cultured in both types of medium were used as a positive control. The impact of the variously modified PLLA foils and/or differences in the composition of the medium were studied with reference to cell adhesion, growth and differentiation. We observed similar adhesion and growth of ADSCs on all PLLA samples when they were cultured in the non-differentiation medium. The differentiation medium supported the expression of specific early, mid-term and/or late markers of differentiation (i.e. type I collagen, αSMA, calponin, smoothelin, and smooth muscle myosin heavy chain) in ADSCs on all tested samples. Moreover, ADSCs cultured in the differentiation medium revealed significant differences in cell growth among the samples that were similar to the differences observed in the cultures of VSMCs. The round morphology of the VSMCs indicated worse adhesion to pristine PLLA, and this sample was also characterized by the lowest cell proliferation. Culturing VSMCs in the differentiation medium inhibited their metabolic activity and reduced the cell numbers. Both cell types formed the most stable monolayer on plasma-treated PLLA and on the PS control. The behaviour of ADSCs and VSMCs on the tested PLLA foils differed according to the specific cell type and culture conditions. The suitable biocompatibility of both cell types on the tested PLLA foils seems to be favourable for vascular tissue engineering purposes.

Cellulose Mesh with Charged Nanocellulose Coatings as a Promising Carrier of Skin and Stem Cells for Regenerative Applications.

Engineering artificial skin constructs is an ongoing challenge. An ideal material for hosting skin cells is still to be discovered. A promising candidate is low-cost cellulose, which is commonly fabricated in the form of a mesh and is applied as a wound dressing. Unfortunately, the structure and the topography of current cellulose meshes are not optimal for cell growth. To enhance the surface structure and the physicochemical properties of a commercially available mesh, we coated the mesh with wood-derived cellulose nanofibrils (CNFs). Three different types of mesh coatings are proposed in this study as a skin cell carrier: positively charged cationic cellulose nanofibrils (cCNFs), negatively charged anionic cellulose nanofibrils (aCNFs), and a combination of these two materials (c+aCNFs). These cell carriers were seeded with normal human dermal fibroblasts (NHDFs) or with human adipose-derived stem cells (ADSCs) to investigate cell adhesion, spreading, morphology, and proliferation. The negatively charged aCNF coating significantly improved the proliferation of both cell types. The positively charged cCNF coating significantly enhanced the adhesion of ADSCs only. The number of NHDFs was similar on the cCNF coatings and on the noncoated pristine cellulose mesh. However, the three-dimensional (3D) structure of the cCNF coating promoted cell survival. The c+aCNF construct proved to combine benefits from both types of CNFs, which means that the c+aCNF cell carrier is a promising candidate for further application in skin tissue engineering.

The Influence of Negative Pressure and of the Harvesting Site on the Characteristics of Human Adipose Tissue-Derived Stromal Cells from Lipoaspirates.

BACKGROUND: Adipose tissue-derived stromal cells (ADSCs) have great potential for cell-based therapies, including tissue engineering. However, various factors can influence the characteristics of isolated ADSCs. METHODS: We studied the influence of the harvesting site, i.e., inner thigh (n = 3), outer thigh (n = 3), outer thigh (n = 3), outer thigh (. RESULTS: We revealed higher initial cell yields from the outer thigh region than from the abdomen region. Negative pressure did not influence the cell yields from the outer thigh region, whereas the yields from the abdomen region were higher under high negative pressure than under low negative pressure. In the subsequent passage, in general, no significant relationship was identified between the different negative pressure and ADSC characteristics. No significant difference was observed in the characteristics of thigh ADSCs and abdomen ADSCs. Only on day 1, the diameter was significantly bigger in outer thigh ADSCs than in abdomen ADSCs. Moreover, we noted a tendency of thigh ADSCs (i.e., inner thigh+outer thigh) to reach a higher cell number on day 7. Discussion. The harvesting site and negative pressure can potentially influence initial cell yields from lipoaspirates. However, for subsequent in vitro culturing and for use in tissue engineering, it seems that the harvesting site and the level of negative pressure do not have a crucial or limiting effect on basic ADSC characteristics.in vitro culturing and for use in tissue engineering, it seems that the harvesting site and the level of negative pressure do not have a crucial or limiting effect on basic ADSC characteristics.

Stem cells: their source, potency and use in regenerative therapies with focus on adipose-derived stem cells - a review.

Stem cells can be defined as units of biological organization that are responsible for the development and the regeneration of organ and tissue systems. They are able to renew their populations and to differentiate into multiple cell lineages. Therefore, these cells have great potential in advanced tissue engineering and cell therapies. When seeded on synthetic or nature-derived scaffolds in vitro, stem cells can be differentiated towards the desired phenotype by an appropriate composition, by an appropriate architecture, and by appropriate physicochemical and mechanical properties of the scaffolds, particularly if the scaffold properties are combined with a suitable composition of cell culture media, and with suitable mechanical, electrical or magnetic stimulation. For cell therapy, stem cells can be injected directly into damaged tissues and organs in vivo. Since the regenerative effect of stem cells is based mainly on the autocrine production of growth factors, immunomodulators and other bioactive molecules stored in extracellular vesicles, these structures can be isolated and used instead of cells for a novel therapeutic approach called "stem cell-based cell-free therapy". There are four main sources of stem cells, i.e. embryonic tissues, fetal tissues, adult tissues and differentiated somatic cells after they have been genetically reprogrammed, which are referred to as induced pluripotent stem cells (iPSCs). Although adult stem cells have lower potency than the other three stem cell types, i.e. they are capable of differentiating into only a limited quantity of specific cell types, these cells are able to overcome the ethical and legal issues accompanying the application of embryonic and fetal stem cells and the mutational effects associated with iPSCs. Moreover, adult stem cells can be used in autogenous form. These cells are present in practically all tissues in the organism. However, adipose tissue seems to be the most advantageous tissue from which to isolate them, because of its abundancy, its subcutaneous location, and the need for less invasive techniques. Adipose tissue-derived stem cells (ASCs) are therefore considered highly promising in present-day regenerative medicine.

Correction: Heritable heading time variation in wheat lines with the same number of Ppd-B1 gene copies.

[This corrects the article DOI: 10.1371/journal.pone.0183745.].

Heritable heading time variation in wheat lines with the same number of Ppd-B1 gene copies.

The ability of plants to identify an optimal flowering time is critical for ensuring the production of viable seeds. The main environmental factors that influence the flowering time include the ambient temperature and day length. In wheat, the ability to assess the day length is controlled by photoperiod (Ppd) genes. Due to its allohexaploid nature, bread wheat carries the following three Ppd-1 genes: Ppd-A1, Ppd-B1 and Ppd-D1. While photoperiod (in)sensitivity controlled by Ppd-A1 and Ppd-D1 is mainly determined by sequence changes in the promoter region, the impact of the Ppd-B1 alleles on the heading time has been linked to changes in the copy numbers (and possibly their methylation status) and sequence changes in the promoter region. Here, we report that plants with the same number of Ppd-B1 copies may have different heading times. Differences were observed among F7 lines derived from crossing two spring hexaploid wheat varieties. Several lines carrying three copies of Ppd-B1 headed 16 days later than other plants in the population with the same number of gene copies. This effect was associated with changes in the gene expression level and methylation of the Ppd-B1 gene.

Evaluation of the potential of chitosan/ß-1,3-glucan/hydroxyapatite material as a scaffold for living bone graft production in vitro by comparison of ADSC and BMDSC behaviour on its surface.

The opinion regarding the origin of adult stem cells that should be used for living bone construct generation is strongly divided in the scientific community. In this study, the potential of chitosan/ß-1,3-glucan/hydroxyapatite (chit/glu/HA) material as a scaffold for bone regeneration applications was evaluated by behaviour comparison of adult stem cells derived from both origins-adipose derived mesenchymal stem cell (ADSC) tissue and bone marrow derived mesenchymal stem cells (BMDSCs). In the case of ADSC isolation, low and high negative pressures were applied during a liposuction procedure in order to determine if negative pressure settings may have an impact on subsequent cell behaviour in vitro. The obtained results demonstrated that the chit/glu/HA material is a promising candidate to be used for living bone graft production in vitro as both ADSCs and BMDSCs revealed a satisfactory proliferation and differentiation ability on its surface. Nevertheless, BMDSCs would be a better choice of adult stem cells since they were better spread, more strongly attached and showed a more superior proliferation and differentiation ability than ADSCs when cultured on the chit/glu/HA scaffold. However, if BMDSCs cannot be isolated, ADSCs may be used for bone construct production but lipoaspirate should be collected under low negative pressure (-200 mm Hg), as high negative pressure (-700 mmHg) applied during liposuction surgery may retard subsequent ADSC proliferation and type I collagen production.