Table of contents

Hydrogels are widely used as reservoirs in drug delivery and scaffolds for tissue engineering. In particular, injectable hydrogel systems, which are formed by physical, chemical, or enzyme-mediated crosslinking reactions in situ, offer the advantages of minimal invasiveness, ease of application, and void-filling property. Examples of these hydrogels are provided in the first part of this paper. In the second part, hydrogels that are formed by the enzymatic activity of horseradish peroxidase (HRP) are highlighted. HRP catalyzes the crosslinking reaction of polymer-phenol conjugates in the presence of hydrogen peroxide (H₂O₂), resulting in hydrogels with tunable gelation rate and crosslinking density. The catalytic mechanism of the HRP-mediated crosslinking reaction is discussed in detail, and the recent biomedical applications of the HRP-crosslinked hydrogels are described. Lastly, the concerns associated with HRP-mediated crosslinking and the future outlook of HRP-crosslinked hydrogels are addressed.

3D biomaterial printing has emerged as a potentially revolutionary technology, promising to transform both research and medical therapeutics. Although there has been recent progress in the field, on-demand fabrication of functional and transplantable tissues and organs is still a distant reality. To advance to this point, there are two major technical challenges that must be overcome. The first is expanding upon the limited variety of available 3D printable biomaterials (biomaterial inks), which currently do not adequately represent the physical, chemical, and biological complexity and diversity of tissues and organs within the human body. Newly developed biomaterial inks and the resulting 3D printed constructs must meet numerous interdependent requirements, including those that lead to optimal printing, structural, and biological outcomes. The second challenge is developing and implementing comprehensive biomaterial ink and printed structure characterization combined with in vitro and in vivo tissue- and organ-specific evaluation. This perspective outlines considerations for addressing these technical hurdles that, once overcome, will facilitate rapid advancement of 3D biomaterial printing as an indispensable tool for both investigating complex tissue and organ morphogenesis and for developing functional devices for a variety of diagnostic and regenerative medicine applications.

Three-dimensional (3D) bioprinting is a disruptive technology for creating organotypic constructs for high-throughput screening and regenerative medicine. One major challenge is the lack of suitable bioinks. Short synthetic self-assembling peptides are ideal candidates. Several classes of peptides self-assemble into nanofibrous hydrogels resembling the native extracellular matrix. This is a conducive microenvironment for maintaining cell survival and physiological function. Many peptides also demonstrate stimuli-responsive gelation and tuneable mechanical properties, which facilitates extrusion before dispensing and maintains the shape fidelity of the printed construct in aqueous media. The inherent biocompatibility and biodegradability bodes well for in vivo applications as implantable tissues and drug delivery matrices, while their short length and ease of functionalization facilitates synthesis and customization. By applying self-assembling peptide inks to bioprinting, the dynamic complexity of biological tissue can be recreated, thereby advancing current biomedical applications of peptide hydrogel scaffolds.

The incorporation of nanomaterials in hydrogels (hydrated networks of crosslinked polymers) has emerged as a useful method for generating biomaterials with tailored functionality. With the available engineering approaches it is becoming much easier to fabricate nanocomposite hydrogels that display improved performance across an array of electrical, mechanical, and biological properties. In this review, we discuss the fundamental aspects of these materials as well as recent developments that have enabled their application. Specifically, we highlight synthesis and fabrication, and the choice of nanomaterials for multifunctionality as ways to overcome current material property limitations. In addition, we review the use of nanocomposite hydrogels within the framework of biomedical and pharmaceutical disciplines.

Biodegradable materials are potentially an advantageous alternative to the traditional metallic fracture fixation devices used in the reconstruction of bone tissue defects. This is due to the occurrence of stress shielding in the surrounding bone tissue that arises from the absence of mechanical stimulus to the regenerating bone due to the mismatch between the elastic modulus of bone and the metal implant. However although degradable polymers may alleviate such issues, these inert materials possess insufficient mechanical properties to be considered as a suitable alternative to current metallic devices at sites of sufficient mechanical loading.

Phosphate based glasses are an advantageous group of materials for tissue regenerative applications due to their ability to completely degrade in vivo at highly controllable rates based on the specific glass composition. Furthermore the release of the glass's constituent ions can evoke a therapeutic stimulus in vivo (i.e. osteoinduction) whilst also generating a bioactive response. The processing of these materials into fibres subsequently allows them to act as reinforcing agents in degradable polymers to simultaneously increase its mechanical properties and enhance its in vivo response.

However despite the various review articles relating to the compositional influences of different phosphate glass systems, there has been limited work summarising the mechanical properties of different phosphate based glass fibres and their subsequent incorporation as a reinforcing agent in degradable composite materials. As a result, this review article examines the compositional influences behind the development of different phosphate based glass fibre compositions intended as composite reinforcing agents along with an analysis of different potential composite configurations. This includes variations in the fibre content, matrix material and fibre architecture as well as other novel composites designs.

We review the details of preparation and of the recently elucidated mechanism of biological (regenerative) activity of a collagen scaffold (dermis regeneration template, DRT) that has induced regeneration of skin and peripheral nerves (PN) in a variety of animal models and in the clinic. DRT is a 3D protein network with optimized pore size in the range 20–125 µm, degradation half-life 14 ± 7 d and ligand densities that exceed 200 µM α1β1 or α2β1 ligands. The pore has been optimized to allow migration of contractile cells (myofibroblasts, MFB) into the scaffold and to provide sufficient specific surface for cell–scaffold interaction; the degradation half-life provides the required time window for satisfactory binding interaction of MFB with the scaffold surface; and the ligand density supplies the appropriate ligands for specific binding of MFB on the scaffold surface. A dramatic change in MFB phenotype takes place following MFB-scaffold binding which has been shown to result in blocking of wound contraction. In both skin wounds and PN wounds the evidence has shown clearly that contraction blocking by DRT is followed by induction of regeneration of nearly perfect organs. The biologically active structure of DRT is required for contraction blocking; well-matched collagen scaffold controls of DRT, with structures that varied from that of DRT, have failed to induce regeneration. Careful processing of collagen scaffolds is required for adequate biological activity of the scaffold surface. The newly understood mechanism provides a relatively complete paradigm of regenerative medicine that can be used to prepare scaffolds that may induce regeneration of other organs in future studies.

Neural stem cells (NSCs) have been a promising candidate for stem cell-based nerve tissue regeneration. Therefore, the design of idea biomaterials that deliver precise regulatory signals to control stem cell fate is currently a crucial issue that depends on a profound understanding of the interactions between NSCs with the surrounding micro-environment. In this work, self-assembled monolayers of alkanethiols on gold with different chemical groups, including hydroxyl (−OH), amino (−NH₂), carboxyl (−COOH) and methyl (−CH₃), were used as a simple model to study the effects of surface chemistry on NSC fate decisions. Contact angle measurement and x-ray photoelectron spectroscopy (XPS) examination implied that all types of alkanethiols self-assembled on gold into a close-packed phase structure with similar molecular densities. In this study, we evaluated NSC adhesion, migration and differentiation in response to different chemical functional groups cultured under serum-free conditions. Our studies showed that NSCs exhibited certain phenotypes with extreme sensitivity to surface chemical groups. Compared with other functional groups, the SAMs with hydroxyl end-groups provided the best micro-environment in promoting NSC migration and maintaining an undifferentiated or neuronal differentiation state.  −NH₂ surfaces directed neural stem cells into astrocytic lineages, while NSCs on  −COOH and  −CH₃ surfaces had a similar potency to differentiate into three nerve lineages. To further investigate the possible signaling pathway, the gene expression of integrin β1 and β4 were examined. The results indicated that a high expression of β1 integrin would probably have a tight correlation with the expression of nestin, which implied the stemness of NSCs, while β4 integrin seemed to correspond to the differentiated NSCs. The results presented here give useful information for the future design of biomaterials to regulate the preservation, proliferation and differentiation of NSCs for central nervous tissue engineering.

Surface/neuron interfaces have played an important role in neural repair including neural prostheses and tissue engineered scaffolds. This comprehensive literature review covers recent studies on the modification of surface/neuron interfaces. These interfaces are identified in cases both where the surfaces of substrates or scaffolds were in direct contact with cells and where the surfaces were modified to facilitate cell adhesion and controlling cell-type specific responses. Different sources of cells for neural repair are described, such as pheochromocytoma neuronal-like cell, neural stem cell (NSC), embryonic stem cell (ESC), mesenchymal stem cell (MSC) and induced pluripotent stem cell (iPS). Commonly modified methods are discussed including patterned surfaces at micro- or nano-scale, surface modification with conducting coatings, and functionalized surfaces with immobilized bioactive molecules. These approaches to control cell-type specific responses have enormous potential implications in neural repair.

Stem cells play essential roles in tissue regeneration in vivo via specific lineage differentiation induced by environmental factors. In the past, biochemical signals were the focus of induced stem cell differentiation. As reported by Engler et al (2006 Cell126 677–89), biophysical signal mediated stem cell differentiation could also serve as an important inducer. With the advancement of material science, it becomes a possible strategy to generate active biophysical signals for directing stem cell fate through specially designed material microstructures. In the past five years, significant progress has been made in this field, and these designed biophysical signals include material elasticity/rigidity, micropatterned structure, extracellular matrix (ECM) coated materials, material transmitted extracellular mechanical force etc. A large number of investigations involved material directed differentiation of mesenchymal stem cells, neural stem/progenitor cells, adipose derived stem cells, hematopoietic stem/progenitor cells, embryonic stem cells and other cells. Hydrogel based materials were commonly used to create varied mechanical properties via modifying the ratio of different components, crosslinking levels, matrix concentration and conjugation with other components. Among them, polyacrylamide (PAM) and polydimethylsiloxane (PDMS) hydrogels remained the major types of material. Specially designed micropatterning was not only able to create a unique topographical surface to control cell shape, alignment, cell–cell and cell–matrix contact for basic stem cell biology study, but also could be integrated with 3D bioprinting to generate micropattered 3D structure and thus to induce stem cell based tissue regeneration. ECM coating on a specific topographical structure was capable of inducing even more specific and potent stem cell differentiation along with soluble factors and mechanical force. The article overviews the progress of the past five years in this particular field.

We are experiencing a new wave of injectable therapeutics (namely/injectable biomaterials) to complement injectable drugs and injectable biologics, and to serve as the basis for injectable combinatorial therapeutics. Injectable biomaterials contribute to the treatment of the fluid-filled defects which often result from disease and injury, by providing the missing physical framework (i.e. the stroma). However, while injectable matrices may be necessary for the successful treatment of certain lesions, they will not likely be sufficient. Chemoattractants for select endogenous cells, or cells themselves, may need to be incorporated into the matrix prior to its injection to ensure the necessary cellular repopulation of the cavitary defect. These agents and others (drugs and biologics) delivered by the matrix represent the new category of injectable combinatorial therapeutics.

Antihyperlipidemic drug statins reportedly promote both bone formation and soft tissue healing. We examined the effect of sustained-release, fluvastatin-impregnated poly(lactic-co-glycolic acid) (PLGA) microspheres on the promotion of bone and gingival healing at an extraction socket in vivo, and the effect of fluvastatin on epithelial cells and fibroblasts in vitro. The maxillary right first molar was extracted in rats, then one of the following was immediately injected, as a single dose, into the gingivobuccal fold: control (no administration), PLGA microspheres without a statin (active control), or PLGA microspheres containing 20 or 40 μg kg⁻¹ of fluvastatin. At days 1, 3, 7, 14, and 28 after injection, bone and soft tissue healing were histologically evaluated. Cell proliferation was measured under the effect of fluvastatin at dosages of 0, 0.01, 0.1, 1.0, 10, and 50 μM. Cell migration and morphology were observed at dosages of 0 and 0.1 μM. Following tooth extraction, the statin significantly enhanced bone volume and density, connective tissue volume, and epithelial wound healing. In the in vitro study, it promoted significant proliferation and migration of epithelial cells and fibroblasts. A single dose of topically administered fluvastatin-impregnated PLGA microspheres promoted bone and soft tissue healing at the extraction site.

Thermally induced phase separation (TIPS) based methods are widely used for the fabrication of porous scaffolds for tissue engineering and related applications. However, formation of a less-/non-porous layer at the scaffold's outer surface at the air–liquid interface, often known as the skin-effect, restricts the cell infiltration inside the scaffold and therefore limits its efficacy. To this end, we demonstrate a TIPS-based process involving the exposure of the just quenched poly(lactide-co-caprolactone):dioxane phases to the pure dioxane for a short time while still being under the quenching strength, herein after termed as the second quenching (2Q). Scanning electron microscopy, mercury intrusion porosimetry and contact angle analysis revealed a direct correlation between the time of 2Q and the gradual disappearance of the skin, followed by the widening of the outer pores and the formation of the fibrous filaments over the surface, with no effect on the internal pore architecture and the overall porosity of scaffolds. The experiments at various quenching temperatures and polymer concentrations revealed the versatility of 2Q in removing the skin. In addition, the in vitro cell culture studies with the human primary fibroblasts showed that the scaffolds prepared by the TIPS based 2Q process, with the optimal exposure time, resulted in a higher cell seeding and viability in contrast to the scaffolds prepared by the regular TIPS. Thus, TIPS including the 2Q step is a facile, versatile and innovative approach to fabricate the polymer scaffolds with a skin-free and fully open porous surface morphology for achieving a better cell response in tissue engineering and related applications.

The repair of bone defects is still a pressing challenge in clinics. Injectable bone cement is regarded as a promising material to solve this problem because of its special self-setting property. Unfortunately, its poor mechanical conformability, unfavorable osteo-genesis ability and insufficient osteo-inductivity seriously limit its clinical application. In this study, novel experimental calcium phosphate silicate bone cement reinforced by carbon fibers (CCPSC) was fabricated and characterized. First, a compressive strength test and cell culture study were carried out. Then, the material was implanted into the femoral epiphysis of beagle dogs to further assess its osteo-conductivity using a micro-computed tomography scan and histological analysis. In addition, we implanted CCPSC into the beagles' intramuscular pouches to perform an elementary investigation of its osteo-inductivity. The results showed that incorporation of carbon fibers significantly improved its mechanical properties. Meanwhile, CCPSC had better biocompatibility to activate cell adhesion as well as proliferation than poly-methyl methacrylate bone cement based on the cell culture study. Moreover, pronounced biodegradability and improved osteo-conductivity of CCPSC could be observed through the in vivo animal study. Finally, a small amount of osteoid was found at the heterotopic site one month after implantation which indicated potential osteo-inductivity of CCPSC. In conclusion, the novel CCPSC shows promise as a bioactive bone substitute in certain load-bearing circumstances.

The aim of the present study was to investigate whether chitosan-based scaffolds modified with D-(+) raffinose and enriched with thiol-modified gelatin could selectively improve osteoblast adhesion and proliferation.

2, 3 and 4.5% chitosan films were prepared. Chitosan suitability for tissue engineering was confirmed by protein adsorption assay. Scaffolds were incubated with a 2.5 mg ml⁻¹ BSA solution and the decrease of protein content in the supernatants was measured by spectrophotometry.

Chitosan films were then enriched with thiol-modified gelatin and their ability to bind BSA was also measured.

Then, 2% chitosan discs with or without thiol-modified gelatin were used as culture substrates for MC3T3-E1 cells. After 72 h cells were stained with trypan blue or with calcein AM and propidium iodide for morphology, viability and proliferation assays. Moreover, cell viability was measured at 48, 72, 96 and 168 h to obtain a growth curve.

Chitosan films efficiently bound and retained BSA proportionally to the concentration of chitosan discs. The amount of protein retained was higher on chitosan enriched with thiol-modified gelatin.

Moreover, chitosan discs allowed the adhesion and the viability of cells, but inhibited their proliferation. The functionalization of chitosan with thiol-modified gelatin enhanced cell spreading and proliferation.

Our data confirm that chitosan is a suitable material for tissue engineering. Moreover, our data show that the enrichment of chitosan with thiol-modified gelatin enhances its biological properties.

Large bone defects are challenging to heal, and often require an osteoconductive and stable support to help the repair of damaged tissue. Bioglass-based scaffolds are particularly promising for this purpose due to their ability to stimulate bone regeneration. However, processing technologies adopted so far do not allow for the synthesis of scaffolds with suitable mechanical properties. Also, conventional sintering processes result in glass de-vitrification, which generates concerns about bioactivity. In this work, we studied the bioactivity and the mechanical properties of Bioglass^® based scaffolds, produced via a powder technology inspired process. The scaffolds showed compressive strengths in the range of 5–40 MPa, i.e. in the upper range of values reported so far for these materials, had tunable porosity, in the range between 55 and 77%, and pore sizes that are optimal for bone tissue regeneration (100–500 μm). We immersed the scaffolds in simulated body fluid (SBF) for 28 d and analyzed the evolution of the scaffold mechanical properties and microstructure. Even if, after sintering, partial de-vitrification occurred, immersion in SBF caused ion release and the formation of a Ca-P coating within 2 d, which reached a thickness of 10–15 μm after 28 d. This coating contained both hydroxyapatite and an amorphous background, indicating microstructural amorphization of the base material. Scaffolds retained a good compressive strength and structural integrity also after 28 d of immersion (6 MPa compressive strength). The decrease in mechanical properties was mainly related to the increase in porosity, caused by its dissolution, rather than to the amorphization process and the formation of a Ca-P coating. These results suggest that Bioglass^® based scaffolds produced via powder metallurgy-inspired technique are excellent candidates for bone regeneration applications.

In recent years, there has been considerable interest in the potential antibacterial properties that bioactive glasses may possess. However, there have been several conflicting reports on the antibacterial efficacy of 45S5 Bioglass^®. Various mechanisms regarding its mode of action have been proposed, such as changes in the environmental pH, increased osmotic pressure, and 'needle-like' sharp glass debris which could potentially damage prokaryotic cell walls and thus inactivate bacteria. In this current study, a systematic investigation was undertaken on the antibacterial efficacy of 45S5 Bioglass^® on Escherichia coli NCTC 10538 and Staphylococcus aureus ATCO 6538 under a range of clinically relevant scenarios including varying Bioglass^® concentration, direct and indirect contact between Bioglass^® and microorganisms, static and shaking incubation conditions, elevated and neutralised pH environments. The results demonstrated that, under elevated pH conditions, Bioglass^® particles have no antibacterial effect on S. aureus while a concentration dependent antibacterial effect against E. coli was observed. However, the antibacterial activity ceased when the pH of the media was neutralised. The results of this current study, therefore, suggest that the mechanism of antibacterial activity of Bioglass^® is associated with changes in the environmental pH; an environment that is less likely to occur in vivo due to buffering of the system.

A porous shape memory scaffold with biomimetic architecture is highly promising for bone tissue engineering applications. In this study, a series of new shape memory polyurethanes consisting of organic poly(ε-caprolactone) (PCL) segments and inorganic polydimethylsiloxane (PDMS) segments in different ratios (9 : 1, 8 : 2 and 7 : 3) was synthesised. These PCL-PDMS copolymers were further engineered into porous fibrous scaffolds by electrospinning. With different ratios of PCL: PDMS, the fibers showed various fiber diameters, thermal behaviour and mechanical properties. Even after being processed into fibrous structures, these PCL-PDMS copolymers maintained their shape memory properties, and all the fibers exhibited excellent shape recovery ratios of  >90% and shape fixity ratios of  >92% after 7 thermo-mechanical cycles. Biological assay results corroborated that the fibrous PCL-PDMS scaffolds were biocompatible by promoting osteoblast proliferation, functionally enhanced biomineralization-relevant alkaline phosphatase expression and mineral deposition. Our study demonstrated that the PCL-PDMS fibers with excellent shape memory properties are promising substrates as bioengineered grafts for bone regeneration.

Recently, cell-based therapies have attracted attention as promising treatments for acute liver failure (ALF). Bone marrow-derived mesenchymal stem cells (MSCs) are potential candidates for co-culture with hepatocytes in poly(lactic acid-glycolic acid) (PLGA) scaffolds to support hepatocellular function. However, the mechanism of culturing protocol using PLGA scaffolds for MSC differentiation into hepatocyte-like cells as well as the therapeutic effect of cell seeded PLGA scaffolds on ALF remain unsatisfactory in clinical application. Here, MSCs and hepatocytes were co-cultured at ratios of 1:2.5 (MSCs: Hep), 1:5 and 1:10, respectively. The proliferation abilities of these co-cultured cells were detected by CCK8, MTT, EdU and by scanning electron microscopy (SEM), and the ability of MSCs to differentiate into hepatocytes was detected by PCR, western blot and immunofluorescence staining. Therapeutic trials of cell seeded PLGA scaffolds were conducted through mouse abdominal cavity transplantation. Results showed that the 1:5 group showed significantly higher cellular proliferation than the 1:2.5 and 1:10 groups, supernatant albumin and urea nitrogen levels were also significantly higher in the 1:5 group than in other two groups. Similarly, the 1:5 group demonstrated better DNA transcription and liver-specific protein (albumin, CK18 and P450) production. Meanwhile, the GalN-stimulated levels of ALT, AST and TBil in mouse serum were down-regulated significantly more by (MSC  +  Hep)-PLGA scaffold treatment than MSC-PLGA or Hep-PLGA scaffold treatments. Furthermore, the (MSC  +  Hep)-PLGA scaffold-treated ALF mice showed a lower immunogenic response level than the other two groups. These data suggested that the ratio of 1:5 (MSC:Hep) co-cultures was the optimal ratio for MSCs to support hepatocellular metabolism and function in PLGA scaffolds in vitro, the (MSC  +  Hep)-PLGA scaffold treatment could perform better restoration for damaged liver function and could give ALF mice a greater survival rate than the monocell seeded PLGA scaffold treatment.

We investigated a Ti6Al4V alloy modified by means of laser peening in the absence of sacrificial coatings. As a consequence of the temperature rise during laser focusing, melting and ablation generated an undulated surface that exhibits an important increase in the content of titanium oxides and OH− ions. Human mesenchymal stem cells and osteoblasts cultured on the oxidized alloy develop noticeable filopodia and lamellipodia. Their paxillin-stained focal adhesions are smaller than in cells attached to the untreated alloy and exhibit a marked loss of colocalization with the ends of actin stress fibers. An important imbalance of phosphorylation and/or dephosphorylation of the focal adhesion kinase is detected in cells grown on the oxidized alloy. Although these mechanisms of adhesion are deeply altered, the surface treatment does not affect cell attachment or proliferation rates on the alloy. Human mesenchymal stem cells cultured on the treated alloy in media containing osteogenic inducers differentiate towards the osteoblastic phenotype to a higher extent than those on the untreated surface. Also, the specific functions of human osteoblasts cultured on these media are enhanced on the treated alloy. In summary, laser peening tailors the Ti6Al4V surface to yield an oxidized layer with increased roughness that allows the colonization and activities of bone-lineage cells.

The acquisition of substantial dermal sealing determines the prognosis of percutaneous titanium-based medical devices or prostheses. A nano-topographic titanium surface with ordered nano-spikes and pores has been shown to induce periodontal-like connective tissue attachment and activate gingival fibroblastic functions. This in vitro study aimed to determine whether an alkali-heat (AH) treatment-created nano-topographic titanium surface could enhance human dermal fibroblastic functions and binding strength to the deposited collagen on the titanium surface.

The surface topographies of commercially pure titanium machined discs exposed to two different AH treatments were evaluated. Human dermal fibroblastic cultures grown on the discs were evaluated in terms of cellular morphology, proliferation, extracellular matrix (ECM) and proinflammatory cytokine synthesis, and physicochemical binding strength of surface-deposited collagen.

An isotropically-patterned, shaggy nano-topography with a sponge-like inner network and numerous well-organized, anisotropically-patterned fine nano-spikes and pores were observed on each nano-topographic surface type via scanning electron microscopy. In contrast to the typical spindle-shaped cells on the machined surfaces, the isotropically- and anisotropically-patterned nano-topographic titanium surfaces had small circular/angular cells containing contractile ring-like structures and elongated, multi-shaped cells with a developed cytoskeletal network and multiple filopodia and lamellipodia, respectively. These nano-topographic surfaces enhanced dermal-related ECM synthesis at both the protein and gene levels, without proinflammatory cytokine synthesis or reduced proliferative activity. Deposited collagen fibers were included in these surfaces and sufficiently bound to the nano-topographies to resist the physical, enzymatic and chemical detachment treatments, in contrast to machined surfaces.

Well-organized, isotropically-/anisotropically-patterned, nano-topographic titanium surfaces with AH treatment-created nano-spikes and pores enhanced human dermal fibroblastic ECM synthesis and established sufficient mechanical integration between the surfaces and ECM to resist various detachment treatments used to experimentally mimic overloading and inflammation.

The manufacture of 3D scaffolds with specific controlled porous architecture, defined microstructure and an adjustable degradation profile was achieved using two-photon polymerization (TPP) with a size of 2  ×  4  ×  2 mm³. Scaffolds made from poly(D,L-lactide-co-ɛ-caprolactone) copolymer with varying lactic acid (LA) and ɛ -caprolactone (CL) ratios (LC16:4, 18:2 and 9:1) were generated via ring-opening-polymerization and photoactivation. The reactivity was quantified using photo-DSC, yielding a double bond conversion ranging from 70% to 90%. The pore sizes for all LC scaffolds were see 300 μm and throat sizes varied from 152 to 177 μm. In vitro degradation was conducted at different temperatures; 37, 50 and 65 °C. Change in compressive properties immersed at 37 °C over time was also measured. Variations in thermal, degradation and mechanical properties of the LC scaffolds were related to the LA/CL ratio. Scaffold LC16:4 showed significantly lower glass transition temperature (T_g) (4.8 °C) in comparison with the LC 18:2 and 9:1 (see 32 °C). Rates of mass loss for the LC16:4 scaffolds at all temperatures were significantly lower than that for LC18:2 and 9:1. The degradation activation energies for scaffold materials ranged from 82.7 to 94.9 kJ mol⁻¹. A prediction for degradation time was applied through a correlation between long-term degradation studies at 37 °C and short-term studies at elevated temperatures (50 and 65 °C) using the half-life of mass loss (Time (M_1/2)) parameter. However, the initial compressive moduli for LC18:2 and 9:1 scaffolds were 7 to 14 times higher than LC16:4 (see 0.27) which was suggested to be due to its higher CL content (20%). All scaffolds showed a gradual loss in their compressive strength and modulus over time as a result of progressive mass loss over time. The manufacturing process utilized and the scaffolds produced have potential for use in tissue engineering and regenerative medicine applications.

Porous titanium has long been desired as a bone substitute material because of its ability to reduce the stress shielding in supporting bone. In order to achieve the various pore structures, we have evolved a moldless process combined with a space holder technique to fabricate porous titanium. This study aims to evaluate which pore size is most suitable for bone regeneration using our process. The mixture comprising Ti powder, wax binder and PMMA spacer was prepared manually at 70 °C which depended on the mixing ratio of each group. Group 1 had an average pore size of 60 μm, group 2 had a maximum pore size of 100 μm, group 3 had a maximum pore size of 200 μm and group 4 had a maximum pore size of 600 μm. These specimens were implanted into rabbit calvaria for three and 20 weeks. Thereafter, histomorphometrical evaluation was performed. In the histomorphometrical evaluation after three weeks, the group with a 600 μm pore size showed a tendency to greater bone ingrowth. However, after 20 weeks the group with a pore size of 100 μm showed significantly greater bone ingrowth than the other groups. This study suggested that bone regeneration into porous titanium scaffolds is pore size-dependent, while bone ingrowth was most prominent for the group with 100 μm-sized pores after 20 weeks in vivo.

Due to high solubility and fast resorption behaviour under physiological conditions, brushite (CaHPO₄⋅2H₂O, calcium monohydrogen phosphate dihydrate, dicalcium phosphate dihydrate) has great potential in bone regeneration applications, both in combination with scaffolds or as a component of calcium phosphate cements. The use of brushite in combination with hydrogels opens up possibilities for new cell-based tissue engineering applications of this promising material. However, published preparation methods of brushite composites, in which the mineral phase is precipitated within the hydrogel network, fail to offer the necessary degree of control over the mineral phase, content and distribution within the hydrogel matrix. The main focus of this study is to address these shortcomings by determining the precise fabrication parameters needed to prepare composites with controlled composition and properties. Composite alginate microbeads were prepared using a counter-diffusion technique, which allows for the simultaneous crosslinking of the hydrogel and precipitation of an inorganic mineral phase. Reliable nucleation of a desired mineral phase within the alginate network proved more challenging than simple aqueous precipitation. This was largely due to ion transport within the hydrogel producing concentration gradients that modified levels of supersaturation and favoured the nucleation of other phases such as hydroxyapatite and octacalcium phosphate, which would otherwise not form. To overcome this, the incorporation of brushite seed crystals resulted in good control during the mineral phase, and by adjusting the number of seeds and amount of precursor concentration, the amount of mineral could be tuned. The material was characterised with a range of physical techniques, including scanning electron microscopy, powder x-ray diffraction and Rietveld refinement, Fourier transform infrared spectroscopy, and thermogravimetric analysis, in order to assess the mineral morphology, phase and amount within the organic matrix. The mineral content of the composite material converted from brushite into hydroxyapatite when submerged in simulated body fluid, indicating possible bioactivity. Additionally, initial cell culture studies revealed that both the material and the synthesis procedure are compatible with cells relevant to bone tissue engineering.

We show that femtosecond laser irradiation of polydimethylsiloxane (PDMS) enables selective and patterned cell growth by altering the wetting properties of the surface associated with chemical and/or topographical changes. In the low pulse energy regime, the surface becomes less hydrophobic and exhibits a low water contact angle compared to the pristine material. X-ray photoelectron spectroscopy (XPS) also reveals an increased oxygen content in the irradiated regions, to which the C2C12 cells and rabbit anti-mouse protein were found to attach preferentially. In the high pulse energy regime, the laser-modified regions exhibit superhydrophobicity and were found to inhibit cell adhesion, whereas cells were found to attach to the surrounding regions due to the presence of nanoscale debris generated by the ablation process.

The use of mucoperiostial flaps during cleft palate surgery is associated with altered palatal bone growth and development. We analyzed the potential usefulness of a bioengineered oral mucosa in an in vivo model of cleft palate. First, a 4 mm palate defect was created in one side of the palate oral mucosa of 3 week-old New Zealand rabbits, and a complete autologous bioengineered oral mucosa (BOM) or acellular fibrin–agarose scaffold (AS) was implanted. No material was implanted in the negative controls (NC), and positive controls were not subjected to palatal defect (PC). Animals were allowed to grow for 6 months and the results were analyzed morphologically (palate mucosa and bone size) and histologically. Results show that palatal mucosa and bone growth and development were significantly altered in NC and AS animals, whereas BOM animals had similar results to PC and the bioengineered oral mucosa was properly integrated in the host palate. The amount and compaction of collagen fibers was similar between BOM and PC, and both groups of animals had comparable contents of proteoglycans and glycoproteins at the palate bone. No differences were found for decorin, osteocalcin and BMP2. The use of bioengineered oral mucosa substitutes is able to improve palate growth and maturation by preventing the alterations found in animals with denuded palate bone. These results support the potential clinical usefulness of BOM substitutes for the treatment of patients with cleft palate and other conditions in which palate mucosa grafts are necessary with consequent bone denudation.

The treatment of large bone defects, particularly those with segmental bone loss, remains a significant clinical challenge as current approaches involving surgery or bone grafting often do not yield satisfactory long-term outcomes. This study reports the evaluation of novel ceramic scaffolds applied as bone graft substitutes in a clinically relevant in vivo model. Baghdadite scaffolds, unmodified or modified with a polycaprolactone coating containing bioactive glass nanoparticles, were implanted into critical-sized segmental bone defects in sheep tibiae for 26 weeks. Radiographic, biomechanical, μ-CT and histological analyses showed that both unmodified and modified baghdadite scaffolds were able to withstand physiological loads at the defect site, and induced substantial bone formation in the absence of supplementation with cells or growth factors. Notably, all samples showed significant bridging of the critical-sized defect (average 80%) with evidence of bone infiltration and remodelling within the scaffold implant. The unmodified and modified baghdadite scaffolds achieved similar outcomes of defect repair, although the latter may have an initial mechanical advantage due to the nanocomposite coating. The baghdadite scaffolds evaluated in this study hold potential for use as purely synthetic bone graft substitutes in the treatment of large bone defects while circumventing the drawbacks of autografts and allografts.

Electrospun nonwovens have been used extensively for tissue engineering applications due to their inherent similarities with respect to fibre size and morphology to that of native extracellular matrix (ECM). However, fabrication of large scaffold constructs is time consuming, may require harsh organic solvents, and often results in mechanical properties inferior to the tissue being treated. In order to translate nonwoven based tissue engineering scaffold strategies to clinical use, a high throughput, repeatable, scalable, and economic manufacturing process is needed. We suggest that nonwoven industry standard high throughput manufacturing techniques (meltblowing, spunbond, and carding) can meet this need. In this study, meltblown, spunbond and carded poly(lactic acid) (PLA) nonwovens were evaluated as tissue engineering scaffolds using human adipose derived stem cells (hASC) and compared to electrospun nonwovens. Scaffolds were seeded with hASC and viability, proliferation, and differentiation were evaluated over the course of 3 weeks. We found that nonwovens manufactured via these industry standard, commercially relevant manufacturing techniques were capable of supporting hASC attachment, proliferation, and both adipogenic and osteogenic differentiation of hASC, making them promising candidates for commercialization and translation of nonwoven scaffold based tissue engineering strategies.

Surface topography and chemistry both play a crucial role on influencing cell response in 3D porous scaffolds in terms of osteogenesis. Inorganic materials with peculiar morphology and chemical functionalities may be proficiently used to improve scaffold properties—in the bulk and along pore surface—promoting in vitro and in vivo osseous tissue in-growth.

The present study is aimed at investigating how bone regenerative properties of composite scaffolds made of poly(Ɛ-caprolactone) (PCL) can be augmented by the peculiar properties of Mg²⁺ ion doped hydroxyapatite (dHA) crystals, mainly emphasizing the role of crystal shape on cell activities mediated by microstructural properties. At the first stage, the study of mechanical response by crossing experimental compression tests and theoretical simulation via empirical models, allow recognizing a significant contribution of dHA shape factor on scaffold elastic moduli variation as a function of the relative volume fraction. Secondly, the peculiar needle-like shape of dHA crystals also influences microscopic (i.e. crystallinity, adhesion forces) and macroscopic (i.e. roughness) properties with relevant effects on biological response of the composite scaffold: differential scanning calorimetry (DSC) analyses clearly indicate a reduction of crystallization heat—from 66.75 to 43.05 J g⁻¹—while atomic force microscopy (AFM) ones show a significant increase of roughness—from (78.15  ±  32.71) to (136.13  ±  63.21) nm—and of pull-off forces—from 33.7% to 48.7%. Accordingly, experimental studies with MG-63 osteoblast-like cells show a more efficient in vitro secretion of alkaline phosphatase (ALP) and collagen I and a more copious in vivo formation of new bone trabeculae, thus suggesting a relevant role of dHA to support the main mechanisms involved in bone regeneration.

We developed a photo-crosslinkable hydrogel-encapsulated three-dimensional (3D) microwell array for studying embryonic stem (ES) cell-derived neuronal differentiation. ES cells were cultured for 5 d in microwells and were subsequently encapsulated by photo-crosslinkable gelatin methacrylate (GelMA) and polyethylene glycol (PEG) hydrogels for an additional 7 d. We observed that ES cells cultured in PEG microwells became uniform-sized embryoid bodies (EBs) compared to those in GelMA microwells. Although ES cells were encapsulated by photo-crosslinkable GelMA and PEG hydrogels, they were highly viable. We demonstrated that uniform-sized EBs encapsulated by GelMA hydrogels in PEG microwells are largely differentiated into neuronal cells. It was revealed that neurites at the periphery of EBs in PEG microwells largely extended into the interface between GelMA hydrogels and PEG microwells for generating neuronal networks. Therefore, this photo-crosslinkable GelMA hydrogel-encapsulated PEG microwell array could be a potentially powerful tool for neurodegenerative disease applications.

Bone healing requires two critical mechanisms, angiogenesis and osteogenesis. In order to improve bone graft substitutes, both mechanisms should be addressed simultaneously. While the individual effects of various bioinorganics have been studied, an understanding of the combinatorial effects is lacking. Cobalt and fluoride ions, in appropriate concentrations, are known to individually favor the vascularization and mineralization processes, respectively. This study investigated the potential of using a combination of fluoride and cobalt ions to simultaneously promote osteogenesis and angiogenesis in human mesenchymal stromal cells (hMSCs). Using a two-step biomimetic method, wells of tissue culture plates were coated with a calcium phosphate (CaP) layer without or with the incorporation of cobalt, fluoride, or both. In parallel, hMSCs were cultured on uncoated well plates, and cultured with cobalt and/or fluoride ions within the media. The results revealed that cobalt ions increased the expression of angiogenic markers, with the effects being stronger when the ions were added as a dissolved salt in cell medium as compared to incorporation into CaP. Cobalt ions generally suppressed the ALP activity, the expression of osteogenic genes, and the level of mineralization, regardless of delivery method. Fluoride ions, individually or in combination with cobalt, significantly increased the expression of many of the selected osteogenic markers, as well as mineral deposition. This study demonstrates an approach to simultaneously target the two essential mechanisms in bone healing: angiogenesis and osteogenesis. The incorporation of cobalt and fluoride into CaPs is a promising method to improve the biological performance of fully synthetic bone graft substitutes.

As novel, promising, man-made nanomaterials with extraordinary properties, carbon nanotubes have been attracting massive attention in regenerative medicine. However, published reports on their potential cytotoxic effects are not concordant and are even conflicting. In the current study, the cytotoxic effects of carboxyl-modified multi-walled carbon nanotubes (COOH-MWCNTs), as well as their influences on the cell adhesion of NIH-3T3 fibroblasts, were thoroughly investigated. Live/dead cell viability assay and cell counting kit-8 assay both indicated that the viability of the NIH-3T3 cells exposed to COOH-MWCNTs in the culture medium was dependent on the latter's concentration. Cell viability increased at COOH-MWCNT concentrations below 50 μg ml⁻¹ and then decreased with increasing concentration. Scanning electron microscopy and immunofluorescent staining of the NIH-3T3 cells revealed that the cells were well adherent to the substrate after exposure to the COOH-MWCNTs for 48 h. Western blot demonstrated that COOH-MWCNT exposure enhanced the expression of adhesion-associated proteins compared with normal cells, peaking at an intermediate concentration. Our study showed that the cytotoxicity of COOH-MWCNTs, as well as their effects on NIH-3T3 fibroblast adhesion, was dose dependent. Therefore, COOH-MWCNT concentrations in the cell culture medium should be considered in the biomedical application of COOH-MWCNTs.