Computational Insights into the Interplay of Mechanical Forces in Angiogenesis.

This study employs a meshless computational model to investigate the impacts of compression and traction on angiogenesis, exploring their effects on vascular endothelial growth factor (VEGF) diffusion and subsequent capillary network formation. Three distinct initial domain geometries were defined to simulate variations in endothelial cell sprouting and VEGF release. Compression and traction were applied, and the ensuing effects on VEGF diffusion coefficients were analysed. Compression promoted angiogenesis, increasing capillary network density. The reduction in the VEGF diffusion coefficient under compression altered VEGF concentration, impacting endothelial cell migration patterns. The findings were consistent across diverse simulation scenarios, demonstrating the robust influence of compression on angiogenesis. This computational study enhances our understanding of the intricate interplay between mechanical forces and angiogenesis. Compression emerges as an effective mediator of angiogenesis, influencing VEGF diffusion and vascular pattern. These insights may contribute to innovative therapeutic strategies for angiogenesis-related disorders, fostering tissue regeneration and addressing diseases where angiogenesis is crucial.

A Comprehensive Look at In Vitro Angiogenesis Image Analysis Software.

One of the complex challenges faced presently by tissue engineering (TE) is the development of vascularized constructs that accurately mimic the extracellular matrix (ECM) of native tissue in which they are inserted to promote vessel growth and, consequently, wound healing and tissue regeneration. TE technique is characterized by several stages, starting from the choice of cell culture and the more appropriate scaffold material that can adequately support and supply them with the necessary biological cues for microvessel development. The next step is to analyze the attained microvasculature, which is reliant on the available labeling and microscopy techniques to visualize the network, as well as metrics employed to characterize it. These are usually attained with the use of software, which has been cited in several works, although no clear standard procedure has been observed to promote the reproduction of the cell response analysis. The present review analyzes not only the various steps previously described in terms of the current standards for evaluation, but also surveys some of the available metrics and software used to quantify networks, along with the detection of analysis limitations and future improvements that could lead to considerable progress for angiogenesis evaluation and application in TE research.

Bioengineering Composite Aerogel-Based Scaffolds That Influence Porous Microstructure, Mechanical Properties and In Vivo Regeneration for Bone Tissue Application.

Tissue regeneration of large bone defects is still a clinical challenge. Bone tissue engineering employs biomimetic strategies to produce graft composite scaffolds that resemble the bone extracellular matrix to guide and promote osteogenic differentiation of the host precursor cells. Aerogel-based bone scaffold preparation methods have been increasingly improved to overcome the difficulties in balancing the need for an open highly porous and hierarchically organized microstructure with compression resistance to withstand bone physiological loads, especially in wet conditions. Moreover, these improved aerogel scaffolds have been implanted in vivo in critical bone defects, in order to test their bone regeneration potential. This review addresses recently published studies on aerogel composite (organic/inorganic)-based scaffolds, having in mind the various cutting-edge technologies and raw biomaterials used, as well as the improvements that are still a challenge in terms of their relevant properties. Finally, the lack of 3D in vitro models of bone tissue for regeneration studies is emphasized, as well as the need for further developments to overcome and minimize the requirement for studies using in vivo animal models.

Interactions between Dental MSCs and Biomimetic Composite Scaffold during Bone Remodeling Followed by In Vivo Real-Time Bioimaging.

Oral-maxillofacial tumor removal can generate critical bone defects and major problems for patients, causing dysfunctionalities and affecting oral competencies such as mastication, swallowing, and breathing. The association of novel biomaterials and cell therapies in tissue engineering strategies could offer new strategies to promote osteomucosa healing. This study focused on the development of a bioengineered construct loaded with human dental follicle cells (MSCs). To increase the bioconstruct integration to the surrounding tissue, a novel and comprehensive approach was designed combining an injectable biomimetic hydrogel and dental stem cells (hDFMSCs) expressing luminescence/fluorescence for semi-quantitative tissue imaging in live animals. This in vivo model with human MSCs was based on an intramembranous bone regeneration process (IMO). Biologically, the biocomposite based on collagen/nanohydroxyapatite filled with cell-loaded osteopontin-fibrin hydrogel (Coll/nanoHA OPN-Fb) exhibited a high cellular proliferation rate, increased bone extracellular matrix deposition (osteopontin) and high ALP activity, indicating an early osteogenic differentiation. Thus, the presence of human OPN enhanced hDFMSC adhesion, migration, and spatial distribution within the 3D matrix. The developed 3D bioconstruct provided the necessary pro-regenerative effect to modulate the biological response, precisely fitting the bone defect with fine-tuned adjustment to the surrounding original structure and promoting oral osteomucosa tissue regeneration. We were also able to track the cells in vivo and evaluate their behavior (migration, proliferation, and differentiation), providing a glimpse into bone regeneration and helping in the optimization of patient-specific therapies.

Full physicochemical and biocompatibility characterization of a supercritical CO2 sterilized nano-hydroxyapatite/chitosan biodegradable scaffold for periodontal bone regeneration.

Despite bone's innate self-renewal capability, some periodontal pathologic and traumatic defects' size inhibits full spontaneous regeneration. This current research characterized a 3D porous biodegradable nano-hydroxyapatite/chitosan (nHAp/CS, 70/30) scaffold for periodontal bone regeneration, which preparation method includes the final solvent extraction and sterilization through supercritical CO2 (scCO2). Micro-CT analysis revealed the fully interconnected porous microstructure of the nHAp/CS scaffold (total porosity 78 %, medium pore size 200 µm) which is critical for bone regeneration. Scanning electron microscopy (SEM) showed HAp crystals forming on the surface of the nHAp/CS scaffold after 21 days in simulated body fluid, demonstrating its bioactivity in vitro. The presence of nHAp in the scaffolds promoted a significantly lower biodegradation rate compared to a plain CS scaffold in PBS. Dynamic mechanical analysis confirmed their viscoelasticity, but the presence of nHAp significantly enhanced the storage modulus (42.34 ± 6.09 kPa at 10 Hz after 28 days in PBS), showing that it may support bone ingrowth at low-load bearing bone defects. Both scaffold types significantly inhibited the growth, attachment and colony formation abilities of S. aureus and E. coli, enhancing the relevance of chitosan in the grafts' composition for the naturally contaminated oral environment. At SEM and laser scanning confocal microscopy, MG63 cells showed normal morphology and could adhere and proliferate inside the biomaterials' porous structure, especially for the nHAp/CS scaffold, reaching higher proliferative rate at day 14. MG63 cells seeded within nHAp/CS scaffolds presented a higher expression of RUNX2, collagen A1 and Sp7 osteogenic genes compared to the CS samples. The in vivo subcutaneous implantation in mice of both scaffold types showed lower biodegradability with the preservation of the scaffolds porous structure that allowed the ingrowth of connective tissue until 5 weeks. Histology shows an intensive and progressive ingrowth of new vessels and collagen between the 3rd and the 5th week, especially for the nHAp/CS scaffold. So far, the scCO2 method enabled the production of a cost-effective and environment-friendly ready-to-use nHAp/CS scaffold with microstructural, chemical, mechanical and biocompatibility features that make it a suitable bone graft alternative for defect sites in an adverse environment as in periodontitis and peri-implantitis.

Translational Research for Orthopedic Bone Graft Development.

Designing biomaterials for bone-substitute applications is still a challenge regarding the natural complex structure of hard tissues. Aiming at bone regeneration applications, scaffolds based on natural collagen and synthetic nanohydroxyapatite were developed, and they showed adequate mechanical and biological properties. The objective of this work was to perform and evaluate a scaled-up production process of this porous biocomposite scaffold, which promotes bone regeneration and works as a barrier for both fibrosis and the proliferation of scar tissue. The material was produced using a prototype bioreactor at an industrial scale, instead of laboratory production at the bench, in order to produce an appropriate medical device for the orthopedic market. Prototypes were produced in porous membranes that were e-beam irradiated (the sterilization process) and then analysed by scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), dynamic mechanical analysis (DMA), cytotoxicity tests with mice fibroblasts (L929), human osteoblast-like cells (MG63) and human MSC osteogenic differentiation (HBMSC) with alkaline phosphatase (ALP) activity and qPCR for osteogenic gene expression. The prototypes were also implanted into critical-size bone defects (rabbits' tibia) for 5 and 15 weeks, and after that were analysed by microCT and histology. The tests performed for the physical characterization of the materials showed the ability of the scaffolds to absorb and retain water-based solvents, as well as adequate mechanical resistance and viscoelastic properties. The cryogels had a heteroporous morphology with microporosity and macroporosity, which are essential conditions for the interaction between the cells and materials, and which consequently promote bone regeneration. Regarding the biological studies, all of the studied cryogels were non-cytotoxic by direct or indirect contact with cells. In fact, the scaffolds promoted the proliferation of the human MSCs, as well as the expression of the osteoblastic phenotype (osteogenic differentiation). The in vivo results showed bone tissue ingrowth and the materials' degradation, filling the critical bone defect after 15 weeks. Before and after irradiation, the studied scaffolds showed similar properties when compared to the results published in the literature. In conclusion, the material production process upscaling was optimized and the obtained prototypes showed reproducible properties relative to the bench development, and should be able to be commercialized. Therefore, it was a successful effort to harness knowledge from the basic sciences to produce a new biomedical device and enhance human health and wellbeing.

Bioengineered Fluorescent Nanoprobe Conjugates for Tracking Human Bone Cells: In Vitro Biocompatibility Analysis.

Herein, we validated novel functionalized hybrid semiconductor bioconjugates made of fluorescent quantum dots (QD) with the surface capped by chitosan (polysaccharide) and chemically modified with O-phospho-L-serine (OPS) that are biocompatible with different human cell sources. The conjugation with a directing signaling molecule (OPS) allows preferential accumulation in human bone mesenchymal stromal cells (HBMSC). The chitosan (Chi) shell with the fluorescent CdS core was characterized by spectroscopical (UV spectrophotometry and photoluminescence), by morphological techniques (Transmission Electron Microscopy (TEM)) and showed small size (ø 2.3 nm) and a stable photoluminescence emission band. The in vitro biocompatibility results were not dependent on the polysaccharide chain length (Chi with higher and lower molecular weight) but were remarkably affected by the surface modification (Chi or Chi-OPS). In addition, the efficiency of nanoparticles uptake by the cells was dependent on cells nature (human primary cells or cell lines) and tissue source (bone or skin) in the presence or absence of the OPS modification. The complex cellular uptake pathways involved in the cell labeling with the nanoparticles do not interfere on the normal cellular biology (adhesion and proliferation), osteogenic differentiation, and gene expression. The bone cells particles uptake evaluation showed a possible pathway by Caveolin-1 that regulates cell transduction in the membrane's Caveolae. Caveolae mediates non-specific endocytosis, and it is upregulated in HBMSC. The OPS-modified nanoparticles promoted an intense intracellular trafficking by the HBMSCs that showed late-osteoblast phenotype with an increase of extracellular matrix (ECM) mineralization (Alizarin red and Von Kossa staining for calcium phosphate crystals). In this work, the OPS modified bioconjugated QD proved to be a reliable and stable fluorescent bioprobe for cell imaging and targeting research that could also help in clarifying some cellular mechanisms of particles intracellular traffic through the cytoplasmic membrane and osteogenic differentiation induction. The in vitro HBMSC's biocompatibility responses indicated that the OPS-modified chitosan QDs have a prospective future in laboratory and pre-clinical applications such as bioimaging analysis and for ex-vivo cellular evaluation of biomedical implants.

Clarifying the Tooth-Derived Stem Cells Behavior in a 3D Biomimetic Scaffold for Bone Tissue Engineering Applications.

Massive amounts of cell are needed for creating tissue engineered 3D constructs, which often requires culture on scaffolds under dynamic conditions to facilitate nutrients and oxygen diffusion. Dynamic cultures are expected to improve cell viability and proliferation rate, when compared to static conditions. However, cells from distinct types and/or tissues sources may respond differently to external stimuli and be incompatible with culture under mechanical shear stress. The first aim of this work was to show that dental stem cells are a valuable source for improving bone regeneration potential of artificial grafts. Mesenchymal stem/stromal cells (MSCs) were isolated from human dental follicle (hDFMSC) and pulp tissues (hDPMSC) and shown to express prototypical stem cell markers. The follicle and pulp dental MSCs capacity to differentiate into osteoblast lineage was evaluated after seeding on 3D porous scaffolds of collagen-nanohydroxyapatite/phosphoserine biocomposite cryogel with osteogenic factors in the culture medium. Both tooth-derived MSCs were able to show high ALP activity, express osteogenic gene markers and secrete osteopontin (OPN). Thereafter, designed multicompartment holder adaptable to spinner flasks was used for dynamic culture (50 rpm) of both dental MSCs types within the porous 3D scaffolds. Standard static culture conditions were used as control. Culture under dynamic conditions promoted follicle MSCs proliferation, while improving their spatial distribution within the scaffold. Under dynamic conditions, the biocomposite scaffold promoted MSCs osteogenic differentiation, as suggested by increased alkaline phosphatase (ALP) activity, higher osteogenic gene expression and OPN deposition. In a similar manner, under dynamic conditions, dental pulp MSCs also showed higher ALP activity and proliferation rate, but lower amounts of osteopontin secretion, when compared to static conditions. After implantation, dental follicle MSCs-loaded 3D scaffolds cultured under dynamic conditions showed higher tissue ingrowth and osteogenic differentiation (higher human OPN secretion) than dental pulp cells. Overall, this study explored the use of tooth-derived stem cells as a clinical alternative source for bone tissue engineering, together with an innovative device for dynamic culture of cell-laden 3D scaffolds. Results showed that human MSCs response upon culture on 3D scaffolds, depends on the cells source and the culture regimen. This suggests that both the type of cells and their culture conditions should be carefully adjusted according to the final clinical application.

Biomimetic Composite Scaffold With Phosphoserine Signaling for Bone Tissue Engineering Application.

In guided bone tissue engineering, successful ingrowth of MSCs depends primarily on the nature of the scaffold. It is well-known that only seconds after implantation, biomaterials are coated by a layer of adsorbed proteins/peptides which modulates the subsequent cell/scaffold interactions, especially at early times after implantation. In this work, nanohydroxyapatite and collagen based composite materials (Coll/nanoHA) were modified with phosphorylated amino acid (O-phospho-L-serine-OPS) to mimic bone tissue, and induce cell differentiation. The choice for this phosphorylated amino acid is due to the fact that osteopontin is a serine-rich glycol-phosphoprotein and has been associated to the early stages of bone formation, and regeneration. Several concentrations of OPS were added to the Coll/nanoHA scaffold and physico-chemical, mechanical, and in vitro cell behavior were evaluated. Afterwards, the composite scaffold with stronger mechanical and best cellular behavior was tested in vivo, with or without previous in vitro culture of human MSC's (bone tissue engineering). The OPS signaling of the biocomposite scaffolds showed similar cellular adhesion and proliferation, but higher ALP enzyme activity (HBMSC). In vivo bone ectopic formation studies allowed for a thorough evaluation of the materials for MSC's osteogenic differentiation. The OPS-scaffolds results showed that the material could modulated mesenchymal cells behavior in favor of osteogenic differentiation into late osteoblasts that gave raised to their ECM with human bone proteins (osteopontin) and calcium deposits. Finally, OPS-modified scaffolds enhanced cell survival, engraftment, migration, and spatial distribution within the 3D matrix that could be used as a cell-loaded scaffold for tissue engineering applications and accelerate bone regeneration processes.

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 type's 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.

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 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.

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.

Injectable biodegradable polycaprolactone-sebacic acid gels for bone tissue engineering.

Tissue engineering constitutes a promising alternative technology to transplantation medicine by creating viable substitutes for failing tissues or organs. The ability to manipulate and reconstitute tissue function has tremendous clinical implications and will most likely play a key role in cell and gene therapies in the coming years. In the present work, a novel injectable and biodegradable biomaterial is reported that could be injected on the human body with a surgical syringe. The material prepared is a blend of polycaprolactone (PCL), a biodegradable and elastic biomedical polymer, and sebacic acid, a natural polymer part of castor oil with low molecular weight to accelerate the slow degradation rate of PCL. The biocompatibility of the blend was evaluated in vitro and its in vivo behavior was also assessed through subcutaneous and bone implantation in rats to evaluate its tissue-forming ability and degradation rate. The results allowed the conclusion that the gel is biocompatible, promotes the differentiation of mesenchymal stem cells, and presents an adequate degradation rate for use in bone tissue engineering. In vivo the gel blends promoted tissue regeneration and adverse reactions were not observed on subcutaneous and bone implants.

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%).