Development of a dextrin-based hydrogel for bone regeneration

[Excerpt] Bone tissue engineering is a very challenging and promising field, which handles with the limitations of bone regenerative capacity and the failure of current orthopedic implants [1]. This work describes the preparation and characterization of an injectable dextrin-based hydrogel (oDex) able to incorporate nanoparticles, cells, biomolecules or Bonelike~ granules [2]. (...)

Biomimetic supramolecular designs for the controlled release of growth factors in bone regeneration

The extracellular matrix (ECM) of tissues is an assembly of insoluble macromolecules that specifically interact with soluble bioactive molecules and regulate their distribution and availability to cells. Recapitulating this ability has been an important target in controlled growth factor delivery strategies for tissue regeneration and requires the design of multifunctional carriers. This review describes the integration of supramolecular interactions on the design of delivery strategies that encompass self-assembling and engineered affinity components to construct advanced biomimetic carriers for growth factor delivery. Several glycan- and peptide-based self-assemblies reported in the literature are highlighted and commented upon. These examples demonstrate how molecular design and chemistry are successfully employed to create versatile multifunctional molecules which self-assemble/disassemble in a precisely predicted manner, thus controlling compartmentalization, transport and delivery. Finally, we discuss whether recent advances in the design and preparation of supramolecular delivery systems have been sufficient to drive real translation towards a clinical impact. 

Silica nanoparticles as dexamethasone delivery systems able to induce the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

Bioactive glasses, especially silica-based materials, are reported to pres- ent osteoconductive and osteoinductive properties, fundamental char- acteristics in bone regeneration [1,2]. Additionally, dexamethasone (Dex) is one of the bioactive agents able to induce the osteogenic differ- entiation of mesenchymal stem cells by increasing the alkaline phos- phatase activity, and the expression levels of Osteocalcin and Bone Sialoprotein [3]. Herein, we synthesised silica (SiO2) nanoparticles (that present inherent bioactivity and ability to act as a sustained drug delivery system), and coated their surface using poly-L-lysine (PLL) and hyaluronic acid (HA) using the layer-by-layer processing technique. Further on, we studied the influence of these new SiO2-polyelectrolyte coated nanoparticles as Dex sustained delivery systems. The SiO2 nanoparticles were loaded with Dex (SiO2-Dex) and coated with PLL and HA (SiO2-Dex-PLL-HA). Their Dex release profile was evaluated and a more sustained release was obtained with the SiO2-Dex-PLL-HA. All the particles were cultured with human bone marrow-derived mes- enchymal stem cells (hBMSCs) under osteogenic differentiation culture conditions. hBMSCs adhered, proliferated and differentiated towards the osteogenic lineage in the presence of SiO2 (DLS 174nm), SiO2-Dex (DLS 175nm) and SiO2-Dex-PLL-HA (DLS 679nm). The presence of these materials induced the overexpression of osteogenic transcripts, namely of Osteocalcin, Bone Sialoprotein and Runx2. Scanning Elec- tron Microscopy/Electron Dispersive Spectroscopy analysis demon- strated that hBMSCs synthesised calcium phosphates when cultured with SiO2-Dex and SiO2-Dex-PLL-HA nanoparticles. These results indi- cate the potential use of these SiO2-polyelectrolytes coated nanoparti- cles as dexamethasone delivery systems capable of promoting osteogenic differentiation of hBMSCs.

Bone turnover markers for early detection of fracture healing disturbances: A review of the scientific literature

Imaging techniques are the standard method for assessment of fracture healing processes. However, these methods are perhaps not entirely reliable for early detection of complications, the most frequent of these being delayed union and non-union. A prompt diagnosis of such disorders could prevent prolonged patient distress and disability. Efforts should be directed towards the development of new technologies for improving accuracy in diagnosing complications following bone fractures. The variation in the levels of bone turnover markers (BTMs) have been assessed with regard to there ability to predict impaired fracture healing at an early stage, nevertheless the conclusions of some studies are not consensual. In this article the authors have revised the potential of BTMs as early predictors of prognosis in adult patients presenting traumatic bone fractures but who did not suffer from osteopenia or postmenopausal osteoporosis. The available information from the different studies performed in this field was systematized in order to highlight the most promising BTMs for the assessment of fracture healing outcome.

Development of advanced cell/tissue culture systems, based on enhanced polymeric scaffolds and sophisticated bioreactors, for tissue engineering applications

Programa Doutoral em Engenharia Biomédica

PHB-HV 3D Scaffold supports osteoblast growth

Bone tissue engineering requires a biocompatible scaffold that supports cell growth and enhances the native repair process. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV) is a biodegradable 3D scaffold with 88.1 â 0.3% porosity and pore size of 163.5 â 0.1 mm. Previous studies demonstrated the potential of PHB-HV as a scaffold in spinal cord repair. The aim of this study was to evaluate PHB-HV as a scaffold for bone regeneration by assessing the cytocompatability of this scaffold.

Semipermeable capsules wrapping a multifunctional and self-regulated co-culture microenvironment for osteogenic differentiation

A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the â stem cell nicheâ , the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

Acellular Gellan-gum based bilayered structures for the regeneration of osteochondral defects: a preclinical study

 In orthopaedics, the management and treatment of osteochondral (OC) defects remains an ongoing clinical challenge. Autologous osteochondral mosaicplasty has been used as a valid option for OC treatments although donor site morbidity remains a source of concern [1]. Engineering a whole structure capable of mimicking different tissues (cartilage and subchondral bone) in an integrated manner could be a possible approach to regenerate OC defects. In our group we have been proposing the use of bilayered structures to regenerate osteochondral defects [2,3]. The present study aims to investigate the pre-clinical performance of bilayered hydrogels and spongy-like hydrogels in in vivo  models (mice and rabbit, respectively), in both subcutaneous and orthotopic models. The bilayered structures were produced from Low Acyl Gellan Gum (LAGG) from Sigma-Aldrich, USA. Cartilage-like layers were obtained from a 2wt% LAGG solution. The bone-like layers were made of 2wt% LAGG with incorporation of hydroxyapatite at 20% and 30% (w/v). Hydrogels and spongy-like were subcutaneouly implanted in mice to evaluate the inflammatory response. Then, OC defects were induced in rabbit knee to create a critical size defect (4 mm diameter and 5 mm depth), and then hydrogels and sponges implanted. Both structures followed different processing methods. The hydrogels were injected allowing in situ  crosslinking. Unlike, the spongy-like were pre-formed by freeze-drying. The studies concerning subcutaneous implantation and critical size OC defect were performed for 2 and 4 weeks time, respectively. Cellular behavior and inflammatory responses were assessed by means of histology staining and biochemical function and matrix deposition by immunohistochemistry. Additionally, both OC structures stability and new cartilage and bone formation were evaluated by using vivo- computed tomography (Scanco 80). The results showed no acute inflammatory response for both approaches. New tissue formation and integration in the adjacent tissues were also observed, which present different characteristic behaviors when comparing hydrogels and sponges response. As future insights, a novel strategy for regeneration of OC defects can be designed encompassing both, hydrogels and spongy-like structures and cellular approaches. References: 1. Espregueira-Mendes J. et al. Osteochondral transplantation using autografts from the upper tibio-fibular joint for the treatment of knee cartilage lesions. Knee Surgery, Sports Traumatology, Arthroscopy 20,1136, 2012. 2. Oliveira JM. et al, Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications: Scaffold design and its performance when seeded with goat bone marrow stromal cells. Biomaterials 27, 6123, 2006. 3. Pereira D R. et al. Gellan Gum-Based Hydrogel Bilayered Scaffolds for Osteochondral Tissue Engineering. Key Engineering Materials 587, 255, 2013.

Highly functional and biodegradable nanofibrous scaffolds combined with stem cells for bone and cartilage tissue engineering

Among the various possible embodiements of Advanced Therapies and in particular of Tissue Engineering the use of temporary scaffolds to regenerate tissue defects is one of the key issues. The scaffolds should be specifically designed to create environments that promote tissue development and not merely to support the maintenance of communities of cells. To achieve that goal, highly functional scaffolds may combine specific morphologies and surface chemistry with the local release of bioactive agents. Many biomaterials have been proposed to produce scaffolds aiming the regeneration of a wealth of human tissues. We have a particular interest in developing systems based in nanofibrous biodegradable polymers1,2. Those demanding applications require a combination of mechanical properties, processability, cell-friendly surfaces and tunable biodegradability that need to be tailored for the specific application envisioned. Those biomaterials are usually processed by different routes into devices with wide range of morphologies such as biodegradable fibers and meshes, films or particles and adaptable to different biomedical applications. In our approach, we combine the temporary scaffolds populated with therapeutically relevant communities of cells to generate a hybrid implant. For that we have explored different sources of adult and also embryonic stem cells. We are exploring the use of adult MSCs3, namely obtained from the bone marrow for the development autologous-based therapies. We also develop strategies based in extra-embryonic tissues, such as amniotic fluid (AF) and the perivascular region of the umbilical cord4 (Whartonâ s Jelly, WJ). Those tissues offer many advantages over both embryonic and other adult stem cell sourcess. These tissues are frequently discarded at parturition and its extracorporeal nature facilitates tissue donation by the patients. The comparatively large volume of tissue and ease of physical manipulation facilitates the isolation of larger numbers of stem cells. The fetal stem cells appear to have more pronounced immunomodulatory properties than adult MSCs. This allogeneic escape mechanism may be of therapeutic value, because the transplantation of readily available allogeneic human MSCs would be preferable as opposed to the required expansion stage (involving both time and logistic effort) of autologous cells. Topics to be covered: This talk will review our latest developments of nanostructured-based biomaterials and scaffolds in combination with stem cells for bone and cartilage tissue engineering.

Chitosan-based scaffolds for tissue regeneration: preparation and microstructure characterization

Scaffolds are porous three-dimensional supports, designed to mimic the extracellular environment and remain temporarily integrated into the host tissue while stimulating, at the molecular level, specific cellular responses to each type of body tissues. The major goal of the research work entertained herein was to study the microstructure of scaffolds made from chitosan (Ch), blends of chitosan and sodium alginate (Ch/NaAlg), blends of chitosan, sodium alginate and calcium chloride (Ch/NaAlg/CaCl2) and blends of chitosan, sodium alginate and hydroxyapatite (Ch/NaAlg/HA). Scaffolds possessing ideal physicochemical properties facilitate cell proliferation and greatly increase the rate of recovery of a damaged organ tissue. Using CT three-dimensional images of the scaffolds, it was observed that all scaffolds had a porosity in the range 64%-92%, a radius of maximum pore occurrence in the range 95m-260m and a permeability in the range 1×10-10-18×10-10 m2. From the results obtained, the scaffolds based on Ch, Ch/NaAlg and Ch/NaAlg/CaCl2 would be most appropriate both for the growth of osteoid and for bone tissue regeneration, while the scaffold made with a blend of Ch/NaAlg/HA, by possessing larger pores size, might be used as a support for fibrovascular tissue.

Development of biomimetic microengineered hydrogel fibers for tendon regeneration

Musculoskeletal diseases are one of the leading causes of disability worldwide. Tendon injuries are responsible for substantial morbidity, pain and disability. Tissue engineering strategies aim at translating tendon structure into biomimetic materials. The main goal of the present study is to develop microengineered hydrogel fibers through the combination of microfabrication and chemical interactions between oppositely charged polyelectrolytes. For this, methacrylated hyaluronic acid (MeHA) and chondroitin sulfate (MeCS) were combined with chitosan (CHT). Hydrogel fibers were obtained by injecting polymer solutions (either MeHA or MeHA/MeCS and CHT) in separate microchannels that join at a y-junction, with the materials interacting upon contact at the interface. To evaluate cell behavior, human tendon derived cells (hTDCs) were isolated from tendon surplus samples during orthopedic surgeries and seeded on top of the fibers. hTDCs adhered to the surface of the fibers, remaining viable, and were found to be expressing CD44, the receptor for hyaluronic acid. The synthesis of hydrogel fibers crosslinkable through both physical and chemical mechanisms combined with microfabrication technology allows the development of biomimetic structures with parallel fibers being formed towards the replication of tendon tissue architecture.

Tendon regeneration through a scaffold-free approach: development of tenogenic magnetic hASCs sheets

Tendon's regeneration is limited, demanding for cell-based strategies to fully restore their functionality upon injury. The concept of magnetic force-based TE(1), generally using magnetic nanoparticles may enable, for example, stem cell stimulation and/or remote control over TE constructs. Thus, we originally propose the development of magnetic cell sheets (magCSs) with tenogenic capability, aimed at promoting tendon's regeneration. A Tenomodulin (TNMD+) subpopulation was sorted from human adipose stem cells (hASCs), using TNMD-coated immunomagnetic beads(2) and used as cell source for the development of magCSs. Briefly, cells were labeled with iron oxide composite particles (Micromod) and cultured for 7 days in α-MEM medium with or without magnetic stimulation provided by a magnetic device (nanoTherics). CSs were retrieved from the plates using magnet attraction as contiguous sheets of cells within its own deposited ECM.

Xenogeneic M2-polarization of Macrophages by undifferentiated and differentiated adipose tissue-derived stem cells

Mesenchymal stem cells (MSCs) are considered to be â â immunologically privileged.â â In a previous work when human adipose tissue-derived stem cells (hASCs) subcutaneously implanted in mice we did not identify an adverse host response1. Recently, it was shown that tissue regeneration could benefit from the polarization of M2 macrophages subpopulations 2. In this study we hypothesised that undifferentiated hASCs and derived osteoblasts and chondrocytes are able to switch murine bone marrow-derived macrophages (mBMMÃ s) into M2 phenotype, aiding tissue regeneration. Murine BMMÃ s were plated in direct contact with undifferentiated and osteo or chondro-differentiated hASCs for 4 h, 10 h, 24 h and 72 h. The cytokine profile was analysed by qRT-PCR and the surface markers were detected by flow cytometry. The direct interaction of both cell types was observed by time lapse microscopy. The results showed that mBMMÃ s polarized after contacting tissue culture polystyrene. This M2 phenotype was maintained along the experiment in direct contact with both undifferentiated and osteo or chondro-differentiated hASCs. This was confirmed by the expression of IL-1, IL-10, IL-4, TNF-a and IFN-g (genetic profile) and surface markers (CD206 + + , CD336 + + , MHC II + and CD86 + + ) detection. These data suggest the potential of hASCs in contemporary xenogenic tissue engineering and regenerative medicine strategies, as well as host immune system modulation in autoimmune diseases. 

Saloplastic membranes as green devices for soft tissue regeneration

Implantable devices must exhibit mechanical properties similar to native tissues to promote appropriate cellular behavior and regeneration. Herein, we report a new membrane manufacture method based on the synthesis of polyelectrolyte complexes (PECs) that exhibit saloplasticity, i.e. variable physical-chemistry using salt as a plasticizer. This is a Green Chemistry approach, as PECs generate structures that are stabilized solely by reversible electrostatic interactions, avoiding the use of harmful crosslinkers completely. Furthermore, natural polyelectrolytes - chitosan and alginate - were used. Upon mixing them, membranes were obtained by drying the PECs at 37ºC, yielding compact PECs without resorting to organicsolvents. The plasticizing effect of salt after synthesis was shown by measuring tensile mechanical properties, which were lower when samples were immersed in high ionic strength solutions.Salt was also used during membrane synthesis in different quan- tities (0 M, 0.15 M and 0.5 M in NaCl) yielding structures with no significant differences in morphology and degradation (around 15% after 3 months in lysozyme). However, swelling was higher (about 10x) when synthesized in the presence of salt. In vitro cell studies using L929 fibroblasts showed that cells adhered and proliferated preferentially in membranes fabricated in the presence of salt (i.e. the membranes with lower tensile strength). Structures with physical-chemical properties controlled with precision open a path to tissue engineering strategies depending on fine tuning mechanical properties and cellular adhesion simply by changing ionic strength during membrane manufacture

Bioactive nanocomposite spheres with proved shape memory capability in bone defects

Bioactive glass nanoparticles (BGNPs) promote an apatite surface layer in physiologic conditions that lead to a good interfacial bonding with bone.1 A strategy to induce bioactivity in non-bioactive polymeric biomaterials is to incorporate BGNPs in the polymer matrix. This combination creates a nanocomposite material with increased osteoconductive properties. Chitosan (CHT) is a polymer obtained by deacetylation of chitin and is biodegradable, non-toxic and biocompatible. The combination of CHT and the BGNPs aims at designing biocompatible spheres promoting the formation of a calcium phosphate layer at the nanocomposite surface, thus enhancing the osteoconductivity behaviour of the biomaterial. Shape memory polymers (SMP) are stimuli-responsive materials that offer mechanical and geometrical action triggered by an external stimulus.2 They can be deformed and fixed into a temporary shape which remains stable unless exposed to a proper stimulus that triggers recovery of their original shape. This advanced functionality makes such SMPs suitable to be implanted using minimally invasive surgery procedures. Regarding that, the inclusion of therapeutic molecules becomes attractive.  We propose the synthesis of shape memory bioactive nanocomposite spheres with drug release capability.3   1.  L. L. Hench, Am. Ceram. Soc. Bull., 1993, 72, 93-98. 2.  A. Lendlein and S. Kelch, Angew Chem Int Edit, 2002, 41, 2034-2057. 3.  Ã . J. Leite, S. G. Caridade and J. F. Mano, Journal of Non-Crystalline Solids (in Press)