Hydrogels have been widely used for articular tissue engineering application, due to their controllable biodegradability and high water content mimicking the biological extracellular matrix. However, they often lack the mechanical support and signaling cues needed to properly guide cells. Graphene and its derivatives have recently emerged as promising materials due to their unique mechanical, physical, chemical proprieties [1]. Although not yet widely used for medical applications, preliminary works suggest that both structural and functional properties of polymeric substrates may be enhanced when combined with graphene oxide (GO) [2]. In this work, reinforced 3D GO/alginate (Alg) hydrogels have been realized and the opportunity of tuning hydrogels mechanical properties in relation to the required physiological needs has been investigated.

After preparing GO nanosheets (Sigma Aldrich) aqueous suspension (1 mg/ml) by ultrasonic treatment, alginate (Manugel GMB, FMC Biopolymer) composite solutions were produced (0, 0.5, 2 wt% GO/Alg). Moulds of agarose (1% w/v in CaCl 0,1M) were prepared to allocate GO/Alg solutions and chemically cross-link gels via diffusion (2 hr. at 37 °C).

GO/Alg hydrogels were characterized through optical/ AFM and FTIR analysis. Biocompatibility tests were performed embedding 3T3 fibroblasts (8 millions/ml) in the GO/Alg hydrogels; cell viability was evaluated at different time points up to two weeks with Dead/alive kit.

Gels mechanical proprieties were assessed via Dynamic Mechanical- Analysis (DMA) up to 28 days of culture (with and w/o cells) at different time points. All tests were performed in triplicate and statistical analyisis carried out (Mann–Whitney U test, n=9, p<0,005).

3D composite GO/alginate hydrogels were successfully realized (3 mm height, 5 mm diameter). Cell viability tests showed that the presence of GO does not decrease cell viability, confirming absence of toxicity, at least up to 2% wt GO/Alg. For all time points cell viability was statistically higher in presence of GO, while there was no significant difference between 0.5 wt% and 2 wt% GO/Alg. Hydrogels functionalized with GO exhibit an Elastic modulus about 3 fold higher than the Alg control at T0. After an initial decreasing of the Young Modulus for the all GO/Alg samples, possibly due to a partial degradation of alginate, a drastic recovery was observed up to 28 days of culture only for GO functionalized samples. The mechanical features improvement was neither mediated nor triggered by cells activity.

We successfully realized a natural-based 3D hydrogel nano-functionalized with graphene, where both mechanical and biological properties were successfully improved. The delayed stabilization of GO/Alg mechanical proprieties may be due either to a chemical interplay between GO and alginate matrix or to GO self-assembling processes over time. Future developments will be carried out to decouple the chemical and topological role of GO on the results observed up to now. Moreover, functional tests will be performed to evaluate the GO effects on in vitro cell differentiation for possible articular clinical applications.

Large bone defect repair has always presented a difficult treatment problem. Marrow-derived osteogenic progenitor cells combined with hydroxyapatite (HA) were used for segmental bone reconstruction. The validity of this model has been shown for the repair of bone defects of critical size in large animal models. We used this cell-based therapeutic approach to treat three patients with large bone defects.

The patients were 41, 22 and 16 years old and had large tibial, ulnar and humeral diaphyseal gaps that ranged in size from 3.0 to 28.3 cm³. Marrow samples were harvested from the iliac crest and osteogenic progenitors isolated and expanded “ex vivo”. The expanded cells were then combined with a highly macroporous bioceramic scaffold whose size and shape reflected each individual bony defect. The cell/bioceramic composites were implanted at the lesion sites. External fixation was used to stabilise the grafts.

At present all patients have been followed up for 4–5 years. Already after the first month after surgery an initial integration at the bone/implant interface was evident. Bone formation in the implants, assessed by X-ray, progressed steadily in the follow-up period. Two patients achieved full functional recovery at 6 months after surgery, one patient at 12 months after surgery. The present report shows that large segmental bone reconstruction can be achieved in humans using osteoprogenitor cells. This technique can be improved by a more biodegradable and more biomechanically resistant scaffold use.

Bone marrow is the tissue where hemopoiesis occurs in close contact with the stromal microenvironment which support hemopoietic stem cell growth and differentiation. The bone marrow stroma is composed of a variety of different cell types providing structural and functional support for hemopoiesis: endothelial cells, adipocytes, smooth muscle cells, reticular cells, osteoblasts and stromal fibroblasts. Among these cell types, stromal fibroblasts have a peculiar biologic relevance. They are in fact able to support hemopoiesis, to differentiate towards osteogenic, chondrogenic and adipogenic lineage and to form a bone structure complete of hemopoietic marrow in in vivo assays. Their in vitro clonogenic counterpart is represented by Colony Forming Units-fibroblasts (CFU-f), which in turn give rise to Bone Marrow Stromal Cells (BMSC). In vivo bone formation by BMSC has been strikingly demonstrated and therefore these cells are considered a progenitor compartment for osteoblasts, responsible for the maintenance of bone turnover throughout life.

BMSC can be easily isolated from bone marrow aspirates. Nevertheless, given the low frequency of BMSC in a marrow sample, a step of extensive in vitro expansion is required to obtain a consistent number of cells available for both reconstruction and repair of mesodermally derived tissues. Moreover, their use for gene and cell therapy of skeletal diseases requires the long-lasting engraftment of BMSC endowed with a residual proliferation potential sufficient to sustain the low, but continuous, bone turnover in adulthood. The maintenance of BMSC stemness and the possibility to reprogram their commitment is therefore a field of primary interest given their potential use in regenerative medicine. Cell therapy of bone lesions by ex vivo expanded BMSC is passing from the phase of experimental animal model to the phase of clinical trials. Bone is repaired via local delivery of cells within a scaffold. Extremely appealing is the possibility of using mesenchymal progenitors in the therapy of genetic bone diseases via systemic infusion. Under some conditions where the local microenvironment is either altered (i.e. injury) or under important remodelling processes (i.e. fetal growth), engraftment of stem and progenitor cells seems to be enhanced. A better understanding of the mechanisms controlling BMSC differentiation and engraftment is required for their exploitation in therapy of human diseases. Furthermore, a better understanding of the interactions occurring between BMSC and biomaterials used to deliver cells in vivo will hopefully extend the field of therapeutic applications of mesenchymal progenitors. In this talk we will go through our experimental evidences on: a) influence of signaling molecule; b) transplantation route and engraftment; c) biomaterials.

Growth factors are essential for a number of cellular functions. Our results show that FGF-2 supplemented BMSC primary cultures display better differentiation potential, a higher degree of osteogenicity and undergo an early increase in telomere size followed by a gradual decrease, whereas in control cultures telomere length decreases with increasing population doublings. In conjunction with clonogenic culture conditions, FGF-2 supplementation extends the life-span of BMSC to over 70 doublings and preserves their differentiation potential up to 50 doublings. All together, these data suggest that FGF-2 supplementation in vitro selects for the survival of a particular subset of cells enriched in pluripotent mesenchymal precursors and may be useful to obtain a large number of cells for mesenchymal tissue repair.

BMSC intravenous infusion has been proposed as a means to support the hematopoiesis in Bone Marrow Transplants or as a vehicle for gene therapy. However, it seems that this route of injection leads to engraftment of a small proportion of BMSC. We have transplanted human BMSC transduced with the human erythropoietin gene, either intravenously or subcutaneously in NOD/SCID mice. Efficiency of engraftment was evaluated monitoring the hematocrit levels. Systemic infusion never increased hematocrit levels, whereas subcutaneous transplantation of the same number of cells induced an important increase of the hematocrit for at least two months. To determine whether the transient effect was due to cell loss or to reduction in expression, we recovered the cells implanted into a tridimensional scaffold, after the normalization of the hematocrit, expanded them in vitro, and re-implanted them in a new group of mice. Again the hematocrit levels rose one week after the transplantation. These results demonstrate that ex-vivo expanded human BMSC are not transplantable by systemic infusion, whereas the local implantation into a 3D scaffold allows their long term engraftment.

Biomaterials for bone regeneration should have a suitable structure to allow cell adhesion and an ideal level of vascularisation, a key factor to achieve new bone formation. Furthermore, they have to be informative, driving the cells towards osteogenesis and allowing the deposition of bone extracellular matrix. Our results indicate that BMSC need a mineralized scaffold to initiate bone formation which will occur with an extent proportional to the availability of biomaterial surface.

Aims: The aim of the study is to evaluate the clinical application in veterinary orthopedics of the bone stromal cells loaded on three-dimensional resorbable osteogenic scaffolds.

Methods: On the basis of the results obtained after an experimental study on 54 adult sheep (data in process), the Authors have carried out a clinical study on 9 dogs of different breed, age,sized with the different orthopaedic lesion associated to large bone defects (from 2 to 4,8 cm) (bone cyst of glenoid rime, non-union of the tibia and of the femur, osteosarcoma of the radio and the proximal humerus, lenghtening of the radius, bone large defect of the distal radius).With the local anesthesia performed with 2% lidocaine the marrow samples were collected from the iliac crest two hours before the surgery. The bone marrow nucleated cells were then isolated from the bone marrow by gradient centrifugation and loaded on the scaffold on biomaterial, which size and shape was defined before performing the surgery. The cells separated were added with some drops of thrombin. The material used for the study was Osteostim Skelite resorbable bone graft substitute (manufactured by Millenium Biologic Inc-.Canada) which chemical composition and size facilitates the ingrowth of bone.

X-ray exams were performed immediately after the surgery. Clinical, ultrasound and x-ray exams were performed after 20 days and then every month.

Results: 7 of 9 treated dogs have shown very good clinical and x-ray results.

Conclusions: One of the objective of the study was to use the fresh bone stromal cells (BMSCs) in clinical applications in large bone defects in the dog. The advantages of using the cells are: they don’t need to be expanded in vitro, they preserve their osteogenic potential to form bone and promote the proper integration of the implant with bone and lastly, the technique is easier and the costs are lower. We use a fully resorbable biomaterial with BMSCs to obtain a complete substitution of large bone defects since the final goal is the complete substitution of the biomaterial scaffold with new formed bone. Persistency of biomaterial, in fact, limits the complete integration of the two (old and new) interfaces and may represent a weak spot in functionality when tensions and loads are fully applied to the bone, in spite of a satisfactory surgical recovery.