Scaffold-free high throughput generation of quiescent valvular microtissues.

AIMS: The major challenge of working with valvular interstitial cells in vitro is the preservation or recovery of their native quiescent state. In this study, a biomimetic approach is used which aims to engineer small volume, high quality valve microtissues, having a potential in regenerative medicine and as a relevant 3D in vitro model to provide insights into valve (patho)biology. METHODS AND RESULTS: To form micro-aggregates, porcine valvular interstitial cells were seeded in agarose micro-wells and cultured in medium supplemented with 250µM Ascorbic Acid 2-phosphate for 22days. Histology showed viable aggregates with normal nuclei and without any signs of calcification. Aggregates stained strongly for GAG and collagen I and reticular fibers were present. ECM formation was quantified and showed a significant increase of GAG, elastin and Col I during aggregate culture. Cultivation of VIC in aggregates also promoted mRNA expression of Col I/III/V, elastin, hyaluronan, biglycan, decorin, versican MMP-1/2/3/9 and TIMP-2 compared to monolayer cultured VIC. Phenotype analysis of aggregates showed a significant decrease in α-SMA expression, and an increase in FSP-1 expression at any time point. Furthermore, VIC aggregates did not show a significant difference in OCN, Egr-1, Sox-9 or Runx2 expression. CONCLUSION: In this study high quality valvular interstitial cell aggregates were generated that are able to produce their own ECM, resembling the native valve composition. The applied and completely cell driven 3D approach overcomes the problems of VIC activation in 2D, by downregulating α-SMA expression and stimulating a homeostatic quiescent VIC state.

Complete Static Repopulation of Decellularized Porcine Tissues for Heart Valve Engineering: An in vitro Study.

To date, a completely in vitro repopulated tissue-engineered heart valve has not been developed. This study focused on sequentially seeding 2 cell populations onto porcine decellularized heart valve leaflets (HVL) and pericardia (PER) to obtain fully repopulated tissues. For repopulation of the interstitium, porcine valvular interstitial cells (VIC) and bone marrow-derived mesenchymal stem cells (BM-MSC) or adipose tissue-derived stem cells (ADSC) were used. In parallel, the culture medium was supplemented with ascorbic acid 2-phosphate (AA) and its effect on recolonization was investigated. Subsequently and in order to obtain an endothelial surface layer similar to those in native HVL, valvular endothelial cells (VEC) were seeded onto the scaffolds. It was shown that VIC efficiently recolonized HVL and partially also PER. On the other hand, stem cells only demonstrated limited or no subsurface cell infiltration of HVL and PER. Interestingly, the addition of AA increased the migratory capacity of both stem cell populations. However, this was more pronounced for BM-MSC, and recolonization of HVL appeared to be more efficient than that of PER tissue. VEC were demonstrated to generate a new endothelial layer on HVL and PER. However, scanning microscopy revealed that these endothelial cells were not allowed to fully spread onto PER. This study provided a proof of concept for the future generation of a bioactive tissue-engineered heart valve by showing that bioactive HVL could be generated in vitro within 14 days via complete repopulation of the interstitium with BM-MSC or VIC and subsequent generation of an entirely new endothelium.

Ovarian tissue cryopreservation in female-to-male transgender people: insights into ovarian histology and physiology after prolonged androgen treatment.

Female-to-male transgender people (trans men) are faced with the risk of losing their reproductive potential owing to gender-affirming hormone treatment and genital reconstructive surgery. This observational, prospective cohort study investigates the effect of prolonged androgen therapy on their ovarian histology and fertility preservation perspectives. Hormone serum levels, ovarian histology and cumulus-oocyte complexes (COC) of 40 trans men were analysed at the moment of hysterectomy with bilateral oophorectomy in the context of genital reconstructive surgery after testosterone treatment (58.18 ± 26.57 weeks). In the cortex, most follicles were primordial (68.52% total follicle count) compared with 20.26% intermediate and 10.74%primary follicles. Few secondary follicles (0.46%) and a single antral follicle were found in the sections analysed. In total, 1313 COC were retrieved from the medulla of 35 patients (37.51 ± 33.58 COC per patient). Anti-Müllerian hormone serum levels were significantly correlated with number of COC (Rs 0.787, P < 0.001). After 48 h in-vitro maturation, 34.30% metaphase II oocytes were obtained, with 87.10% having a normal spindle structure. In conclusion, the cortical follicle distribution in trans men, after more than a year of testosterone treatment, seems to be surprisingly normal. This work confirms the presence and in-vitro maturation potential of cumulus-oocyte complexes.

Alternative Routes to Induce Naïve Pluripotency in Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naïve pluripotent state. Efforts have been made to trigger naïve pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naïve genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene-free naïve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naïve hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated naïve pluripotency genes, and exhibited dependence on signaling pathways similar to naïve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs that is fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation.

Redifferentiation of High-Throughput Generated Fibrochondrocyte Micro-Aggregates: Impact of Low Oxygen Tension.

In meniscus tissue engineering strategies, enhancing the matrix quality of the neomeniscal tissue is important. When the differentiated phenotype of fibrochondrocytes is lost, the quality of the matrix becomes compromised. The objective of this study was to produce uniform fibrochondrocyte micro-aggregates with desirable phenotype and tissue homogeneity in large quantities using a simple and reproducible method. Furthermore, we investigated if hypoxia could enhance the matrix quality. Porcine fibrochondrocytes were expanded at 21% oxygen until passage 3 (P3) and a gene expression profile was determined. P3 fibrochondrocytes were cultivated in chondrogenic medium at 5 and 21% oxygen in high-throughput agarose chips containing 2,865 microwells 200 µm in diameter. Evaluation included live/dead staining, histological examination, immunohistochemistry, dimethylmethylene blue assay and real-time reverse transcriptase quantitative polymerase chain reaction of the micro-aggregates. Gene expression analysis showed a drastic decline in collagen II and high expression of collagen I during monolayer culture. After 4 days, uniform and stable micro-aggregates could be produced. The redifferentiation and matrix quality of the hypoxic cultured micro-aggregates were enhanced relative to the normoxic cultures. Sulfated glycosaminoglycan synthesis was significantly higher, and collagen II expression and the collagen II/collagen I ratio were significantly upregulated in the hypoxic cultures. High-throughput production of uniform microtissues holds promise for the generation of larger-scale tissue engineering constructs or optimization of redifferentiation mechanisms for clinical applications.

Cryogel-PCL combination scaffolds for bone tissue repair.

The present work describes the development and the evaluation of cryogel-poly-ε-caprolactone combinatory scaffolds for bone tissue engineering. Gelatin was selected as cell-interactive biopolymer to enable the adhesion and the proliferation of mouse calvaria pre-osteoblasts while poly-ε-caprolactone was applied for its mechanical strength required for the envisaged application. In order to realize suitable osteoblast carriers, methacrylamide-functionalized gelatin was introduced into 3D printed poly-ε-caprolactone scaffolds created using the Bioplotter technology, followed by performing a cryogenic treatment which was concomitant with the redox-initiated, covalent crosslinking of the gelatin derivative (i.e. cryogelation). In a first part, the efficiency of the cryogelation process was determined using gel fraction experiments and by correlating the results with conventional hydrogel formation at room temperature. Next, the optimal cryogelation parameters were fed into the combinatory approach and the scaffolds developed were characterized for their structural and mechanical properties using scanning electron microscopy, micro-computed tomography and compression tests respectively. In a final part, in vitro biocompatibility assays indicated a good colonization of the pre-osteoblasts and the attachment of viable cells onto the cryogenic network. However, the results also show that the cellular infiltration throughout the entire scaffold is suboptimal, which implies that the scaffold design should be optimized by reducing the cryogel density.

Indirect additive manufacturing as an elegant tool for the production of self-supporting low density gelatin scaffolds.

The present work describes for the first time the production of self-supporting low gelatin density (<10 w/v%) porous scaffolds using methacrylamide-modified gelatin as an extracellular matrix mimicking component. As porous scaffolds starting from low gelatin concentrations cannot be realized with the conventional additive manufacturing techniques in the abscence of additives, we applied an indirect fused deposition modelling approach. To realize this, we have printed a sacrificial polyester scaffold which supported the hydrogel material during UV crosslinking, thereby preventing hydrogel structure collapse. After complete curing, the polyester scaffold was selectively dissolved leaving behind a porous, interconnective low density gelatin scaffold. Scaffold structural analysis indicated the success of the selected indirect additive manufacturing approach. Physico-chemical testing revealed scaffold properties (mechanical, degradation, swelling) to depend on the applied gelatin concentration and methacrylamide content. Preliminary biocompatibility studies revealed the cell-interactive and biocompatible properties of the materials developed.

Plasma surface modification of polylactic acid to promote interaction with fibroblasts.

In this work, medium pressure plasma treatment of polylactic acid (PLA) is investigated. PLA is a biocompatible aliphatic polymer, which can be used for bone fixation devices and tissue engineering scaffolds. Due to inadequate surface properties, cell adhesion and proliferation are far less than optimal and a surface modification is required for most biomedical applications. By using a dielectric barrier discharge (DBD) operating at medium pressure in different atmospheres, the surface properties of a PLA foil are modified. After plasma treatment, water contact angle measurements showed an increased hydrophilic character of the foil surface. X-ray photoelectron spectroscopy (XPS) revealed an increased oxygen content. Cell culture tests showed that plasma modification of PLA films increased the initial cell attachment both quantitatively and qualitatively. After 1 day, cells on plasma-treated PLA showed a superior cell morphology in comparison with unmodified PLA samples. However, after 7 days of culture, no significant differences were observed between untreated and plasma-modified PLA samples. While plasma treatment improves the initial cell attachment, it does not seem to influence cell proliferation. It has also been observed that the difference between the 3 discharge gases is negligible when looking at the improved cell-material interactions. From economical point of view, plasma treatments in air are thus the best choice.

An optimized growth factor cocktail for ovine mesenchymal stem cells.

Growth factors that regulate proliferation, migration, and invasion of ovine mesenchymal stem cells (oMSCs) are not well defined. In this study, we have evaluated five growth factors for their ability to initiate and support in vitro proliferation, migration, and invasion of oMSCs. oMSCs were exposed to different doses and combinations of the growth factors: basic fibroblast growth factor (bFGF), transforming growth factor-ß (TGF-ß), epidermal growth factor (EGF), insulin growth factor-I (IGF-I), connective tissue growth factor, and platelet-derived growth factor-AB (PDGF-AB). Cellular proliferation, motility, and invasiveness were assayed. The most proliferative stimulating growth factors are PDGF-AB+TGF-ß and PDGF-AB+IGF-I. Combinations EGF+bFGF and EGF+bFGF+PDGF-AB demonstrated the greatest ability to stimulate migration. Moreover, the triple cocktail EGF+bFGF+TGF-ß has the most significant effect on invasion. Different growth factor cocktails are required to enhance proliferation, migration, and invasion. These results may be useful for the development of a tissue-engineered heart valve by stimulating cellular repopulation.

Static secondary ion mass spectrometry for the surface characterisation of individual nanofibres of polycaprolactone functionalised with an antibacterial additive.

Electrospinning (ES) of polymer solutions generates non-woven webs of nanofibres. The fibre diameter ranges between 10 nm and 1 µm depending on the operating conditions. Surface functionalisation can be performed by the use of suitable additives. Detailed characterisation of the molecular composition at the fibre surface is a key issue. Biodegradable nanowebs with potential antibacterial activity have been prepared by ES of solutions containing polycaprolactone (PCL) and a functionalising additive with PCL segments and hexyldimethylammonium groups (PCLhexaq). Static secondary ion mass spectrometry with Bi(3)(+) projectiles has been applied to individual nanofibres. The positive ion mass spectra contain several signals with high structural specificity allowing the presence of PCLhexaq to be traced back in spite of its low concentration (0.16-1.4% w/w relative to PCL) and its structural similarity to the PCL fibre matrix. Imaging of structural ions visualises the homogeneous distribution of PCLhexaq over the fibre surface. Quantifying the surface concentration of PCLhexaq relative to that of PCL reveals electric field-driven surface enrichment of the additive during ES. Finally, nanofibres subjected to leaching in water for up to 72 h have been analysed. The PCLhexaq surface concentration decreases almost linearly with time at a rate of 0.6% h(-1).

Efficient differentiation of human embryonic stem cells into a homogeneous population of osteoprogenitor-like cells.

The use of human embryonic stem cells (hESC) in both research and therapeutic applications requires relatively large homogeneous populations of differentiated cells. The differentiation of three hESC lines into highly homogeneous populations of osteoprogenitor-like (hESC-OPL) cells is reported here. These cells could be expanded in a defined culture system for more than 18 passages, and showed a fibroblast-like morphology and a normal stable karyotype. The cells were strongly positive for the same antigenic markers as mesenchymal stem cells but negative for markers of haematopoetic stem cells. The hESC-OPL cells were able to differentiate into the osteogenic, but not into the chondrogenic or adipogenic, lineage and were positive for markers of early stages of osteogenic differentiation. When cultured in the presence of osteogenic supplements, the cells indicated the capacity to achieve, under inductive conditions, a mature osteoblast phenotype. The differentiation protocol is based on a monolayer approach, and does not require any exogenous factors other than fetal calf serum, or coculture systems of animal or human origin. This method is likely to be amenable to large-scale production of homogeneous osteoprogenitor-like cells and thus overcomes one of the major problems of differentiation of hESC, with important relevance for further cell therapy studies.

Effects of different sterilization methods on the physico-chemical and bioresponsive properties of plasma-treated polycaprolactone films.

For most tissue engineering applications, surface modification and sterilization of polymers are critical aspects determining the implant success. The first part of this study is thus dedicated to modifying polycaprolactone (PCL) surfaces via plasma treatment using a medium pressure dielectric barrier discharge, while the second part focuses on the sterilization of plasma-modified PCL. Chemical and physical surface changes are examined making use of water contact angle goniometry (WCA), x-ray photoelectron spectroscopy and atomic force microscopy. Bioresponsive properties are evaluated by performing cell culture tests. The results show that air and argon plasmas decrease the WCA significantly due to the incorporation of oxygen-containing functionalities onto the PCL surface, without modifying its morphology. Extended treatment times lead to PCL degradation, especially in the case of air plasma. In addition to surface modification, the plasma potential to sterilize PCL is studied with appropriate treatment times, but sterility has not been achieved so far. Therefore, plasma-modified films are subjected to UV, H2O2 plasma (HP) and ethylene oxide (EtO) sterilizations. UV exposure of 3 h does not alter the PCL physico-chemical properties. A decreased wettability is observed after EtO sterilization, attributable to the modification of PCL chain ends reacting with EtO molecules. HP sterilization increases the WCA of the plasma-treated samples, presumably due to the scission of the hydrophilic bonds generated during the prior plasma treatments. Moreover, HP modifies the PCL surface morphology. For all the sterilizations, an improved cell adhesion and proliferation is observed on plasma-treated films compared to untreated ones. EtO shows the lowest proliferation rate compared to HP and UV. Overall, of the three sterilizations, UV is the most effective, since the physical alterations provoked by HP might interfere with the structural integrity when it comes to 3D scaffolds, and the chemical modifications caused by EtO, in addition to its toxicity, interfere with PCL bioactivity.

Impact of Detergent-Based Decellularization Methods on Porcine Tissues for Heart Valve Engineering.

To date an optimal decellularization protocol of heart valve leaflets (HVL) and pericardia (PER) with an adequate preservation of the extracellular matrix (ECM) is still lacking. This study compares a 4 day Triton X-100-based protocol with faster SDC-based protocols for the decellularization of cardiac tissues. Decellularized and non-treated HVL and PER were processed for histological, biochemical and mechanical analysis to determine the effect of these agents on the structure, ECM components, and biomechanical properties. Tissues treated with SDC-based protocols still showed nuclear material, whereas tissues treated with Triton X-100 1% + ENZ ± TRYP were completely cell free. For both decellularized tissues, an almost complete washout of glycosaminoglycans, a reduction of soluble collagen and an alteration of the surface ultrastructure was observed. Interestingly, only the elastic fibers of pericardial tissue were affected and this tissue had a decreased maximum load. This study showed that both detergents had a similar impact on the ECM. However, Triton X-100 1% +DNase/RNase (ENZ) ± Trypsin (TRYP) is the only protocol that generated completely cell free bioscaffolds. Also, our study clearly demonstrated that the decellularization agents have more impact on pericardial tissues than on heart valve leaflets. Thus, for the purpose of tissue engineering of heart valves, it is advisable to use valvular rather than pericardial matrices.

Autologous plasma and its supporting role in fat graft survival: A relevant vector to counteract resorption in lipofilling.

Fat grafting has become a widespread technique for different reconstructive and esthetic purposes. However, the disadvantage of fat grafting is the unpredictable resorption rate that often necessitates repetitive procedures, which in turn may have an impact on the morbidity. During the immediate, post-graft, ischemic period, cells survive due to the process of plasmatic imbibition. This biological phenomenon precedes the ingrowth of neo-capillaries that eventually nourish the graft and help establish a long-term homeostatic equilibrium. Both partners, the graft and the recipient bed, contribute to the revascularization process. Hypothetically, enrichment of the recipient site with autologous plasma could have a beneficial role to enhance fat graft survival. We investigated whether plasma supported the viability of the lipoaspirate (LA) material. Plasma was isolated from blood samples collected from eight patients during the elective lipofilling procedures. An in vitro study assessed the viability of LA cells using plasma as a culture medium compared to the traditional culture media. In vitro analysis confirmed sustained viability of LA cells compared to the standard media and control media during 7 consecutive days. The behavior of the fat grafts in plasma showed similarities with those incubated in the traditional culture media. In future, these findings could be translated to a clinical setting. Plasma is the only autologous substrate available in large quantities in the human body. The addition of the supporting agents, such as plasma, could contribute to a better graft survival with more stable clinical outcomes in the long term. The rationale behind the technique is based on the phenomenon of plasmatic imbibition and the reasoning that the extracellular matrix plays a pivotal role in cellular survival.

Galactose-functionalized gelatin hydrogels improve the functionality of encapsulated HepG2 cells.

The present study investigates the effect of galactosylated gelatin on encapsulated HepG2 cells. Methacrylamide modified gelatin is evaluated and compared with its galactosylated counterpart with respect to effects on viability, morphological characteristics, proliferation, and the expression of hepatocyte specific markers. The research reveals that further modifications of methacrylamide modified gelatin are possible without affecting the survival of the encapsulated cells (viability of 90%). Moreover, the study demonstrates a clear and long-term (up to 21 d) improvement in hepatocyte specific gene expression when the cells are encapsulated in the galactosylated gelatin. It is concluded that the use of galactosylated gelatin derivates supports the hepatocyte phenotype.

High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

Enzymatic mineralization of silk scaffolds.

The present study focuses on the alkaline phosphatase (ALP) mediated formation of apatitic minerals on porous silk fibroin protein (SFP) scaffolds. Porous SFP scaffolds impregnated with different concentrations of ALP are homogeneously mineralized under physiological conditions. The mineral structure is apatite while the structures differ as a function of the ALP concentration. Cellular adhesion, proliferation, and colonization of osteogenic MC3T3 cells improve on the mineralized SFP scaffolds. These findings suggest a simple process to generate mineralized scaffolds that can be used to enhanced bone tissue engineering-related utility.

Injectable self-gelling composites for bone tissue engineering based on gellan gum hydrogel enriched with different bioglasses.

Hydrogels of biocompatible calcium-crosslinkable polysaccharide gellan gum (GG) were enriched with bioglass particles to enhance (i) mineralization with calcium phosphate (CaP); (ii) antibacterial properties and (iii) growth of bone-forming cells for future bone regeneration applications. Three bioglasses were compared, namely one calcium-rich and one calcium-poor preparation both produced by a sol-gel technique (hereafter referred to as A2 and S2, respectively) and one preparation of composition close to that of the commonly used 45S5 type (hereafter referred to as NBG). Incubation in SBF for 7 d, 14 d and 21 d caused apatite formation in bioglass-containing but not in bioglass-free samples, as confirmed by FTIR, XRD, SEM, ICP-OES, and measurements of dry mass, i.e. mass attributable to polymer and mineral and not water. Mechanical testing revealed an increase in compressive modulus in samples containing S2 and NBG but not A2. Antibacterial testing using biofilm-forming meticillin-resistant staphylococcus aureus (MRSA) showed markedly higher antibacterial activity of samples containing A2 and S2 than samples containing NBG and bioglass-free samples. Cell biological characterization using rat mesenchymal stem cells (rMSCs) revealed a stimulatory effect of NBG on rMSC differentiation. The addition of bioglass thus promotes GG mineralizability and, depending on bioglass type, antibacterial properties and rMSC differentiation.

Biocompatibility properties of surface-modified poly(dimethylsiloxane) for urinary applications.

An electronic sensor system for urinary bladder pressure monitoring requires an imbedding into a biocompatible, flexible, and liquid-impermeable material. Poly(dimethylsiloxane) (PDMS) was selected in the present set-up as packaging material because it fulfills the abovementioned requirements. However, the surface of PDMS is hydrophobic and causes undesired interactions with salts, proteins, and cells present in urine. To reduce possible interactions of urine salts in the urinary bladder, monomers, [2-(methacryloyloxy)ethyl]-dimethyl-3-sulfopropyl-ammonium hydroxide (sulfobetaine) and 2-acrylamido-2-methylpropyl sulfonic acid, were grafted onto the surface through oxygen plasma treatment. A reduction in salt deposition between the pure PDMS and the modified PDMS was observed both in vitro (artificial urine flow over the surface) and in vivo (implants into the urinary bladder of experimental pigs). Additionally, a 10-fold reduction in salt deposition was observed in vitro due to grafting of the monomers onto the surface. These modified PDMS materials proved also to be biocompatible in cell cultures, which was further confirmed by histological screening of the bladder tissue after implantation in an in vivo pig model.

Cell survival and proliferation after encapsulation in a chemically modified Pluronic(R) F127 hydrogel.

Pluronic® F127 is a biocompatible, injectable, and thermoresponsive polymer with promising biomedical applications. In this study, a chemically modified form, i.e., Pluronic ALA-L with tailored degradation rate, was tested as an encapsulation vehicle for osteoblastic cells. UV cross-linking of the modified polymer results in a stable hydrogel with a slower degradation rate. Toxicological screening showed no adverse effects of the modified Pluronic ALA-L on the cell viability. Moreover, high viability of embedded cells in the cross-linked Pluronic ALA-L was observed with life/death fluorescent staining during a 7-day-culture period. Cells were also cultured on macroporous, cross-linked gelatin microbeads, called CultiSpher-S® carriers, and encapsulated into the modified cross-linked hydrogel. Also, in this situation, good cell proliferation and migration could be observed in vitro. Preliminary in vivo tests have shown the formation of new bone starting from the injected pre-loaded CultiSpher-S® carriers.