Christiane L. Salgado

Preparation and characterization of collagen‐nanohydroxyapatite biocomposite scaffolds by cryogelation method for bone tissue engineering applications

Recent efforts of bone repair focus on development of porous scaffolds for cell adhesion and proliferation. Collagen-nanohydroxyapatite (HA) scaffolds (70:30; 50:50; and 30:70 mass percentage) were produced by cryogelation technique using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide as crosslinking agents. A pure collagen scaffold was used as control. Morphology analysis revealed that all cryogels had highly porous structure with interconnective porosity and the nanoHA aggregates were randomly dispersed throughout the scaffold structure. Chemical analysis showed the presence of all major peaks related to collagen and HA in the biocomposites and indicated possible interaction between nanoHA aggregates and collagen molecules. Porosity analysis revealed an enhancement in the surface area as the nanoHA percentage increased in the collagen structure. The biocomposites showed improved mechanical properties as the nanoHA content increased in the scaffold. As expected, the swelling capacity decreased with the increase of nanoHA content. In vitro studies with osteoblasts cells showed that they were able to attach and spread in all cryogels surfaces. The presence of collagen-nanoHA biocomposites resulted in higher overall cellular proliferation compared to pure collagen scaffold. A statistically significant difference between collagen and collagen-nanoHA cryogels was observed after 21 day of cell culture. These innovative collagen-nanoHA cryogels could have potentially appealing application as scaffolds for bone regeneration.

Combinatorial cell–3D biomaterials cytocompatibility screening for tissue engineering using bioinspired superhydrophobic substrates

We report on the development of a new array-based screening flat platform with the potential to be used as a high-throughput device based on biomimetic polymeric substrates for combinatorial cell/3D biomaterials screening assays in the context of tissue engineering. Polystyrene was used to produce superhydrophobic surfaces based on the so-called lotus effect. Arrays of hydrophilic regions could be patterned in such surfaces using UV/ozone radiation, generating devices onto which combinatorial hydrogel spots were deposited. The biological performance of encapsulated cells in hydrogels could be tested in an in vitro 3D environment assuming that each site was isolated from the others due to the high contrast of wettability between the patterned spots and the superhydrophobic surroundings. Three different polymers-chitosan, collagen and hyaluronic acid-were combined with alginate in different proportions in order to obtain combinatorial binary alginate-based polymeric arrays. The effect of the addition of gelatin to the binary structures was also tested. The gels were chemically analyzed by FTIR microscopic mapping. Cell culture results varied according to the hydrogel composition and encapsulated cell types (L929 fibroblast cells and MC3T3-E1 pre-osteoblast cells). Cell viability and number could be assessed by conventional methods, such as MTS reduction test and dsDNA quantification. Non-destructive image analysis was performed using cytoskeleton and nuclei staining agents and the results were consistent with the ones obtained by conventional sample-destructive techniques. Briefly, L929 cells showed higher number and viability for higher alginate-content and collagen-containing hydrogels, while MC3T3-E1 showed higher cell viability and cell number in lower alginate-content and chitosan containing hydrogels. The addition of gelatin did not influence significantly cell metabolic activity or cell number in any of the encapsulated cell types.

Combinatorial on-chip study of miniaturized 3D porous scaffolds using a patterned superhydrophobic platform

One of the main challenges in tissue engineering (TE) is to obtain optimized products, combining biomaterials, cells and soluble factors able to stimulate tissue regeneration. Multiple combinations may be considered by changing the conditions among these three factors. The unpredictable response of each combination requires time-consuming tests. High-throughput methodologies have been proposed to master such complex analyses in TE. Usually, these tests are performed using cells cultured into 2D biomaterials or by dispensing arrays of cell-loaded hydrogels. For the first time an on-chip combinatorial study of 3D miniaturized porous scaffolds is proposed, using a patterned bioinspired superhydrophobic platform. Arrays of biomaterials are dispensed and processed in situ as porous scaffolds with distinct composition, surface characteristics, porosity/pore size, and mechanical properties. On-chip porosity, pore size, and mechanical properties of scaffolds based on chitosan and alginate are assessed by adapting microcomputed tomography equipment and a dynamic mechanical analyzer, as well as cell response after 24 hours. The interactions between cell types of two distinct origins-osteoblast-like and fibroblasts-and the scaffolds modified with fibronectin are studied and validated by comparison with conventional destructive methods (dsDNA quantification and MTS tests). Physical and biological on-chip analyses are coherent with the conventional measures, and conclusions about the most favorable conditions for each cell type are taken.

Biocompatibility and biodegradation of polycaprolactone‐sebacic acid blended gels

Tissue engineering aims at creating biological body parts as an alternative for transplanting tissues and organs. A current new approach for such materials consists in injectable biodegradable polymers. Their major advantages are the ability to fill-in defects, easy incorporation of therapeutic agents or cells, and the possibility of minimal invasive surgical procedures. Polycaprolactone (PCL) is a promising biodegradable and elastic biomaterial, with the drawback of low-degradation kinetics in vivo. In this work a biodegradable injectable gel of PCL blended with sebacic acid (SA) was prepared, to improve the degradation rate of the biomaterial. SA is known for its high degradation rate, although in high concentrations it could originate a pH decrease and thus disturb the biocompatibility of PCL. Degradation tests on phosphate buffered saline were carried out using 5% of SA on the blend and the biomaterial stability was evaluated after degradation using differential scanning calorimetry, dynamical mechanical analysis, and scanning electronic microscopy. After degradation the elastic properties of the blend decreased and the material became more crystalline and stiffer, although at a lower extent when compared with pure PCL. The blend also degraded faster with a loss of the crystalline phase on the beginning (30 days), although its thermal and mechanical properties remained comparable with those of the pure material, thus showing that it achieved the intended objectives. After cell assays the PCL-SA gel was shown to be cytocompatible and capable of maintaining high cell viability (over 90%).

Antibacterial activity and biocompatibility of three-dimensional nanostructured porous granules of hydroxyapatite and zinc oxide nanoparticles—an in vitro and in vivo study

Ceramic scaffolds are widely studied in the bone tissue engineering field due to their potential in regenerative medicine. However, adhesion of microorganisms on biomaterials with subsequent formation of antibiotic-resistant biofilms is a critical factor in implant-related infections. Therefore, new strategies are needed to address this problem. In the present study, three-dimensional and interconnected porous granules of nanostructured hydroxyapatite (nanoHA) incorporated with different amounts of zinc oxide (ZnO) nanoparticles were produced using a simple polymer sponge replication method. As in vitro experiments, granules were exposed to Staphylococcus aureus and Staphylococcus epidermidis and, after 24 h, the planktonic and sessile populations were assessed. Cytocompatibility towards osteoblast-like cells (MG63 cell line) was also evaluated for a period of 1 and 3 days, through resazurin assay and imaging flow cytometry analysis. As in vivo experiments, nanoHA porous granules with and without ZnO nanoparticles were implanted into the subcutaneous tissue in rats and their inflammatory response after 3, 7 and 30 days was examined, as well as their antibacterial activity after 1 and 3 days of S. aureus inoculation. The developed composites proved to be especially effective at reducing bacterial activity in vitro and in vivo for a weight percentage of 2% ZnO, with a low cell growth inhibition in vitro and no differences in the connective tissue growth and inflammatory response in vivo. Altogether, these results suggest that nanoHA-ZnO porous granules have a great potential to be used in orthopaedic and dental applications as a template for bone regeneration and, simultaneously, to restrain biomaterial-associated infections.

Biodegradation, biocompatibility, and osteoconduction evaluation of collagen‐nanohydroxyapatite cryogels for bone tissue regeneration

Designing biomimetic biomaterials inspired by the natural complex structure of bone and other hard tissues is still a challenge nowadays. The control of the biomineralization process onto biomaterials should be evaluated before clinical application. Aiming at bone regeneration applications, this work evaluated the in vitro biodegradation and interaction between human bone marrow stromal cells (HBMSC) cultured on different collagen/nanohydroxyapatite cryogels. Cell proliferation, differentiation, morphology, and metabolic activity were assessed through different protocols. All the biocomposite materials allowed physiologic apatite deposition after incubation in simulated body fluid and the cryogel with the highest nanoHA content showed to have the highest mechanical strength (DMA). The study clearly showed that the highest concentration of nanoHA granules on the cryogels were able to support cell types survival, proliferation, and individual functionality in a monoculture system, for 21 days. In fact, the biocomposites were also able to differentiate HBMSCs into osteoblastic phenotype. The composites behavior was also assessed in vivo through subcutaneous and bone implantation in rats to evaluate its tissue-forming ability and degradation rate. The cryogels Coll/nanoHA (30 : 70) promoted tissue regeneration and adverse reactions were not observed on subcutaneous and bone implants. The results achieved suggest that scaffolds of Coll/nanoHA (30 : 70) should be considered promising implants for bone defects that present a grotto like appearance with a relatively small access but a wider hollow inside. This material could adjust to small dimensions and when entering into the defect, it could expand inside and remain in close contact with the defect walls, thus ensuring adequate osteoconductivity.

Fluorescent bionanoprobes based on quantum dot-chitosan–O-phospho-L-serine conjugates for labeling human bone marrow stromal cells

Bone replacement materials might be promising alternatives to autogenous bone transplants during the repair and reconstruction of human bone tissues. However, bone healing is a very complex process, and the roles of phosphatidylserine (PS) and its moieties are not yet completely understood. In the present study, fluorescent quantum dots (QDs) functionalized with chitosan–O-phospho-L-serine (chi–OPS) conjugates have been synthesized and characterized while focusing on their potential applications as nanoprobes for labeling human bone marrow stromal cells (hBMSC). Essentially, chitosan was covalently linked to the peptide (O-phospho-L-serine, OPS) through the formation of amide bonds. In this sequence, these chi–OPS conjugates were utilized as direct capping ligands during CdS QDs (CdS/chi–OPS) biofunctionalization, which was achieved using a single-step process in an aqueous medium at room temperature. The core–shell nanostructures were characterized in detail by UV-visible spectroscopy (UV-Vis), photoluminescence spectroscopy (PL), atomic force microscopy (AFM), and transmission electron microscopy (TEM) with selected area electron diffraction (SEAD). The TEM images associated with the UV-vis optical absorption results indicated that ultra-small nanocrystals were formed with average diameters ranging from 2.2 to 2.8 nm. In addition, the PL results showed that the nanoconjugates exhibited “green” fluorescent activity under ultraviolet excitation. Cell viability was assessed in vitro via an MTT analysis, revealing that the bioconjugates were not cytotoxic after 3 days of incubation. Moreover, a quantitative flow cytometry (QFC) analysis and confocal fluorescence microscopy (CFA) were performed, verifying the fluorescence-labeling efficiency and the endocytosis of the bionanoprobes by hBMSC. In summary, innovative fluorescent conjugates were developed with properties for use as biomarkers when imaging and detecting bone tissue regeneration and metabolic events.