Enhancing volumetric muscle loss (VML) recovery in a rat model using super durable hydrogels derived from bacteria.

Bacteria can be programmed to deliver natural materials with defined biological and mechanical properties for controlling cell growth and differentiation. Here, we present an elastic, resilient and bioactive polysaccharide derived from the extracellular matrix of Pantoea sp. BCCS 001. Specifically, it was methacrylated to generate a new photo crosslinkable hydrogel that we coined Pantoan Methacrylate or put simply PAMA. We have used it for the first time as a tissue engineering hydrogel to treat VML injuries in rats. The crosslinked PAMA hydrogel was super elastic with a recovery nearing 100 %, while mimicking the mechanical stiffness of native muscle. After inclusion of thiolated gelatin via a Michaelis reaction with acrylate groups on PAMA we could also guide muscle progenitor cells into fused and aligned tubes - something reminiscent of mature muscle cells. These results were complemented by sarcomeric alpha-actinin immunostaining studies. Importantly, the implanted hydrogels exhibited almost 2-fold more muscle formation and 50 % less fibrous tissue formation compared to untreated rat groups. In vivo inflammation and toxicity assays likewise gave rise to positive results confirming the biocompatibility of this new biomaterial system. Overall, our results demonstrate that programmable polysaccharides derived from bacteria can be used to further advance the field of tissue engineering. In greater detail, they could in the foreseeable future be used in practical therapies against VML.

Injectable hydrogels for cartilage and bone tissue regeneration: A review.

Annually, millions of patients suffer from irreversible injury owing to the loss or failure of an organ or tissue caused by accident, aging, or disease. The combination of injectable hydrogels and the science of stem cells have emerged to address this persistent issue in society by generating minimally invasive treatments to augment tissue function. Hydrogels are composed of a cross-linked network of polymers that exhibit a high-water retention capacity, thereby mimicking the wet environment of native cells. Due to their inherent mechanical softness, hydrogels can be used as needle-injectable stem cell carrier materials to mend tissue defects. Hydrogels are made of different natural or synthetic polymers, displaying a broad portfolio of eligible properties, which include biocompatibility, low cytotoxicity, shear-thinning properties as well as tunable biological and physicochemical properties. Presently, novel ongoing developments and native-like hydrogels are increasingly being used broadly to improve the quality of life of those with disabling tissue-related diseases. The present review outlines various future and in-vitro applications of injectable hydrogel-based biomaterials, focusing on the newest ongoing developments of in-situ forming injectable hydrogels for bone and cartilage tissue engineering purposes.

Multi-leveled Nanosilicate Implants Can Facilitate Near-Perfect Bone Healing.

Several studies have shown that nanosilicate-reinforced scaffolds are suitable for bone regeneration. However, hydrogels are inherently too soft for load-bearing bone defects of critical sizes, and hard scaffolds typically do not provide a suitable three-dimensional (3D) microenvironment for cells to thrive, grow, and differentiate naturally. In this study, we bypass these long-standing challenges by fabricating a cell-free multi-level implant consisting of a porous and hard bone-like framework capable of providing load-bearing support and a softer native-like phase that has been reinforced with nanosilicates. The system was tested with rat bone marrow mesenchymal stem cells in vitro and as a cell-free system in a critical-sized rat bone defect. Overall, our combinatorial and multi-level implant design displayed remarkable osteoconductivity in vitro without differentiation factors, expressing significant levels of osteogenic markers compared to unmodified groups. Moreover, after 8 weeks of implantation, histological and immunohistochemical assays indicated that the cell-free scaffolds enhanced bone repair up to approximately 84% following a near-complete defect healing. Overall, our results suggest that the proposed nanosilicate bioceramic implant could herald a new age in the field of orthopedics.

Composite Graded Melt Electrowritten Scaffolds for Regeneration of the Periodontal Ligament-to-Bone Interface.

Periodontitis is a ubiquitous chronic inflammatory, bacteria-triggered oral disease affecting the adult population. If left untreated, periodontitis can lead to severe tissue destruction, eventually resulting in tooth loss. Despite previous efforts in clinically managing the disease, therapeutic strategies are still lacking. Herein, melt electrowriting (MEW) is utilized to develop a compositionally and structurally tailored graded scaffold for regeneration of the periodontal ligament-to-bone interface. The composite scaffolds, consisting of fibers of polycaprolactone (PCL) and fibers of PCL-containing magnesium phosphate (MgP) were fabricated using MEW. To maximize the bond between bone (MgP) and ligament (PCL) regions, we evaluated two different fiber architectures in the interface area. These were a crosshatch pattern at a 0/90° angle and a random pattern. MgP fibrous scaffolds were able to promote in vitro bone formation even in culture media devoid of osteogenic supplements. Mechanical properties after MgP incorporation resulted in an increase of the elastic modulus and yield stress of the scaffolds, and fiber orientation in the interfacial zone affected the interfacial toughness. Composite graded MEW scaffolds enhanced bone fill when they were implanted in an in vivo periodontal fenestration defect model in rats. The presence of an interfacial zone allows coordinated regeneration of multitissues, as indicated by higher expression of bone, ligament, and cementoblastic markers compared to empty defects. Collectively, MEW-fabricated scaffolds having compositionally and structurally tailored zones exhibit a good mimicry of the periodontal complex, with excellent regenerative capacity and great potential as a defect-specific treatment strategy.

Tissue-specific melt electrowritten polymeric scaffolds for coordinated regeneration of soft and hard periodontal tissues.

Periodontitis is a chronic inflammatory condition that often causes serious damage to tooth-supporting tissues. The limited successful outcomes of clinically available approaches underscore the need for therapeutics that cannot only provide structural guidance to cells but can also modulate the local immune response. Here, three-dimensional melt electrowritten (i.e., poly(ε-caprolactone)) scaffolds with tissue-specific attributes were engineered to guide differentiation of human-derived periodontal ligament stem cells (hPDLSCs) and mediate macrophage polarization. The investigated tissue-specific scaffold attributes comprised fiber morphology (aligned vs. random) and highly-ordered architectures with distinct strand spacings (small 250 µm and large 500 µm). Macrophages exhibited an elongated morphology in aligned and highly-ordered scaffolds, while maintaining their round-shape on randomly-oriented fibrous scaffolds. Expressions of periostin and IL-10 were more pronounced on the aligned and highly-ordered scaffolds. While hPDLSCs on the scaffolds with 500 µm strand spacing show higher expression of osteogenic marker (Runx2) over 21 days, cells on randomly-oriented fibrous scaffolds showed upregulation of M1 markers. In an orthotopic mandibular fenestration defect model, findings revealed that the tissue-specific scaffolds (i.e., aligned fibers for periodontal ligament and highly-ordered 500 µm strand spacing fluorinated calcium phosphate [F/CaP]-coated fibers for bone) could enhance the mimicking of regeneration of natural periodontal tissues.

Injectable and adhesive hydrogels for dealing with wounds.

INTRODUCTION: The development of wound dressing materials that combine healing properties, ability to self-repair the material damages, skin-friendly adhesive nature, and competent mechanical properties have surpassing functional importance in healthcare. Due to their specificity, hydrogels have been recognized as a new gateway in biological materials to treat dysfunctional tissues. The design and creation of injectable hydrogel-based scaffolds have extensively progressed in recent years to improve their therapeutic efficacy and to pave the way for their easy minimally invasive administration. Hence, injectable hydrogel biomaterials have been prepared to eventually translate into minimally invasive therapy and pose a lasting effect on regenerative medicine. AREAS COVERED: This review highlights the recent development of adhesive and injectable hydrogels that have applications in wound healing and wound dressing. Such hydrogel materials are not only expected to improve therapeutic outcomes but also to facilitate the easy surgical process in both wound healing and dressing. EXPERT OPINION: Wound healing seems to be an appealing approach for treating countless life-threatening disorders. With the average increase of life expectancy in human societies, an increase in demand for injectable skin replacements and drug delivery carriers for chronic wound healing is expected.

3D-Printed Regenerative Magnesium Phosphate Implant Ensures Stability and Restoration of Hip Dysplasia.

Osteoarthritis of the hip is a painful and debilitating condition commonly occurring in humans and dogs. One of the main causes that leads to hip osteoarthritis is hip dysplasia. Although the current surgical methods to correct dysplasia work satisfactorily in many circumstances, these are associated with serious complications, tissue resorption, and degeneration. In this study, a one-step fabrication of a regenerative hip implant with a patient-specific design and load-bearing properties is reported. The regenerative hip implant is fabricated based on patient imaging files and by an extrusion assisted 3D printing process using a flexible, bone-inducing biomaterial. The novel implant can be fixed with metallic screws to host bone and can be loaded up to physiological loads without signs of critical permanent deformation or failure. Moreover, after exposing the hip implant to accelerated in vitro degradation, it is confirmed that it is still able to support physiological loads even after losing ≈40% of its initial mass. In addition, the osteopromotive properties of the novel hip implant is demonstrated as shown by an increased expression of osteonectin and osteocalcin by cultured human mesenchymal stem cells after 21 days. Overall, the proposed hip implant provides an innovative regenerative and mechanically stable solution for hip dysplasia treatment.

Nanoclay Reinforced Biomaterials for Mending Musculoskeletal Tissue Disorders.

Nanoclay-reinforced biomaterials have sparked a new avenue in advanced healthcare materials that can potentially revolutionize treatment of musculoskeletal defects. Native tissues display many important chemical, mechanical, biological, and physical properties that engineered biomaterials need to mimic for optimal tissue integration and regeneration. However, it is time-consuming and difficult to endow such combinatorial properties on materials via feasible and nontoxic procedures. Fortunately, a number of nanomaterials such as graphene, carbon nanotubes, MXenes, and nanoclays already display a plethora of material properties that can be transferred to biomaterials through a simple incorporation procedure. In this direction, the members of the nanoclay family are easy to functionalize chemically, they can significantly reinforce the mechanical performance of biomaterials, and can provide bioactive properties by ionic dissolution products to upregulate cartilage and bone tissue formation. For this reason, nanoclays can become a key component for future orthopedic biomaterials. In this review, we specifically focus on the rapidly decreasing gap between clinic and laboratory by highlighting their application in a number of promising in vivo studies.

Combinatorial fluorapatite-based scaffolds substituted with strontium, magnesium and silicon ions for mending bone defects.

In bone tissue engineering, ionic doping using bone-related minerals such as magnesium (Mg) or strontium (Sr) is a promising strategy to make up for the inherent disadvantages (low solubility) of various apatite-based materials (such as fluorapatite (FAp) and hydroxyapatite (HA)). Therefore, some studies in recent years have tried to address the lack-of-methodology to improve the properties of bioceramics in the field. Even though the outcome of the studies has shown some promises, the influence of doped elements on the structures and properties of in-vitro and in-vivo mineralized FAp has not been investigated in detail so far. Thus, it is still an open question mark in the field. In this work, strontium modified fluorapatite (Sr-FAp), magnesium and silicon modified fluorapatite (Mg-SiFAp) bioceramics were synthesized using a mechanical alloying methodology. Results showed that the doped elements could decrease the crystallinity of FAp (56%) to less than 45% and 39% for Sr-FAp and Mg-SiFAp, respectively. Moreover, in-vitro studies revealed that Sr-FAp significantly enhanced osteogenic differentiation of hMSCs, after 21 days of culture, compared to Mg-SiFAp at both osteogenic and normal media. Then, in vivo bone formation in a defect of rat femur filled with a Sr-FAp and Mg-SiFAp compared to empty defect was investigated. Histological analysis revealed an increase in bone formation three weeks after implanting Sr-FAp compared to Mg-SiFAp and the empty defect. These results suggest that compared to magnesium and silicon, strontium ion significantly promotes bone formation in fluorapatite, making it appropriate for filling bone defects.

Tough magnesium phosphate-based 3D-printed implants induce bone regeneration in an equine defect model.

One of the important challenges in bone tissue engineering is the development of biodegradable bone substitutes with appropriate mechanical and biological properties for the treatment of larger defects and those with complex shapes. Recently, magnesium phosphate (MgP) doped with biologically active ions like strontium (Sr2+) have shown to significantly enhance bone formation when compared with the standard calcium phosphate-based ceramics. However, such materials can hardly be shaped into large and complex geometries and more importantly lack the adequate mechanical properties for the treatment of load-bearing bone defects. In this study, we have fabricated bone implants through extrusion assisted three-dimensional (3D) printing of MgP ceramics modified with Sr2+ ions (MgPSr) and a medical-grade polycaprolactone (PCL) polymer phase. MgPSr with 30 wt% PCL (MgPSr-PCL30) allowed the printability of relevant size structures (>780 mm3) at room temperature with an interconnected macroporosity of approximately 40%. The printing resulted in implants with a compressive strength of 4.3 MPa, which were able to support up to 50 cycles of loading without plastic deformation. Notably, MgPSr-PCL30 scaffolds were able to promote in vitro bone formation in medium without the supplementation with osteo-inducing components. In addition, long-term in vivo performance of the 3D printed scaffolds was investigated in an equine tuber coxae model over 6 months. The micro-CT and histological analysis showed that implantation of MgPSr-PCL30 induced bone regeneration, while no bone formation was observed in the empty defects. Overall, the novel polymer-modified MgP ceramic material and extrusion-based 3D printing process presented here greatly improved the shape ability and load-bearing properties of MgP-based ceramics with simultaneous induction of new bone formation.

Stable and Antibacterial Magnesium-Graphene Nanocomposite-Based Implants for Bone Repair.

Magnesium (Mg)-based alloys are promising biodegradable materials for bone repair applications. However, due to their rapid degradation and high corrosion rate, Mg-based alloys are typically associated with in vivo infections and implant failure. This study evaluated the synergistic stability and anti-inflammatory properties that could potentially be achieved by the modification of the Mg alloy with graphene nanoparticles (Gr). Incorporation of low dosages of Gr (0.18 and 0.50 wt %) in a Mg alloy with aluminum (Al, 1 wt %) and copper (Cu, 0.25 wt %) was successfully achieved by a spark plasma sintering (SPS) method. Notably, the degradation rate of the Mg-based alloys was reduced approximately 4-fold and the bactericidal activity was enhanced up to 5-fold with incorporation of only 0.18 wt % Gr to the Mg-1Al-Cu matrix. Moreover, the modified Mg-based nanocomposites with 0.18 wt % Gr demonstrated compressive properties within the range of native cancellous bone (modulus of approximately 6 GPa), whereas in vitro studies with human mesenchymal stromal cells (hMSCs) showed high cytocompatibility and superior osteogenic properties compared to non-Gr-modified Mg-1Al-Cu implants. Overall, this study provides foundations for the fabrication of stable, yet fully resorbable, Mg-based bone implants that could reduce implant-associated infections.

Chitosan-heparin nanoparticle coating on anodized NiTi for improvement of blood compatibility and biocompatibility.

The aim of this study was to simultaneously improve blood compatibility and corrosion resistance of nitinol via two-step process; anodizing and consequently coating with chitosans-heparin nanoparticles. Moreover, the role of these surface modification processes on the heparin release kinetic and blood compatibility was investigated. Finally, the interaction between human umbilical vein endothelial cells (HUVECs) and surface modified samples was investigated. Electrochemical characterization revealed that while Ni ions released from the anodized sample (9 ppb), chitosan-heparin nanoparticle coatings prohibited from Ni ion release form NiTi substrate. Moreover, the controlled release of heparin was found from chitosan-heparin nanoparticle coating deposited on the nanotubes, leading to significant improvement of blood compatibility. Finally, HUVECs were attached and proliferated on the chitosan-heparin nanoparticle coated samples confirming the cell compatibility of samples. In summary, results proved that two-step anodizing process and heparin release could promote both endothelial cell compatibility and blood compatibility to nitinol surface which might be appropriate for coronary stent application.

A three-layered hollow tubular scaffold as an enhancement of nerve regeneration potential.

A significant clinical challenge in the surgery of peripheral nervous system injured via accidents and natural disease is development of biomimetic grafts which could potentially promote nerve repair and regeneration. Although various engineered neural tissue scaffolds have been proposed to support the neural cell functions, they have not been able to instantaneously mimic the whole characteristics of endogenous microenvironment. In this study, we proposed a three-layered tubular scaffold which could provide appropriate electrical, mechanical and biological properties for peripheral nerve engineering. While the inter layer was graphene (Gr) embedded alginate-polyvinyl alcohol (AP-Gr) fibrous scaffold with well-defined anisotropy, the outer layer was double network scaffold of polycaprolactone fumarate (PCLF) and eggshell membrane (ESM). These two layers were attached together using a polycaprolactone (PCL) fibrous membrane, a middle layer, via a simple melting process. Results showed that while the electrical conductivity of the three-layered scaffold was similar to that of AP-Gr fibrous layer, the strength of the three-layered scaffold was significantly improved compared to AP-Gr and ESM-PCLF (1.5 and 1.1 times, respectively) attributed to well attachment of the two layers. As a proof-of-concept, PC12 cell attachment, proliferation, and alignment were studied on the developed three-layered scaffold. The majority of the cells (55%) aligned (<20°) along the major axis of fibers features. Furthermore, electrical stimulation revealed positive effect on the alignment of PC12 cells and change in the cell morphology. With the ease of fabrication and mechanical robustness, the three-layered scaffold of AP-Gr and ESM-PCLF might be utilized as a versatile system for the engineering of peripheral nerve tissue.

Hemocompatible and Bioactive Heparin-Loaded PCL-α-TCP Fibrous Membranes for Bone Tissue Engineering.

The combination of bioactive components such as calcium phosphates and fibrous structures are encouraging niche-mimetic keys for restoring bone defects. However, the importance of hemocompatibility of the membranes is widely ignored. Heparin-loaded nanocomposite poly(ε-caprolactone) (PCL)-α-tricalcium phosphate (α-TCP) fibrous membranes are developed to provide bioactive and hemocompatible constructs for bone tissue engineering. Nanocomposite membranes are optimized based on bioactivity, mechanical properties, and cell interaction. Consequently, various concentrations of heparin molecules are loaded within nanocomposite fibrous membranes. In vitro heparin release profiles reveal a sustained release of heparin over the period of 14 days without an initial burst. Moreover, heparin encapsulation enhances mesenchymal stem cell (MSC) attachment and proliferation, depending on the heparin content. It is concluded that the incorporation of heparin within TCP-PCL fibrous membranes provides the most effective cellular interactions through synergistic physical and chemical cues.

Anisotropic architecture and electrical stimulation enhance neuron cell behaviour on a tough graphene embedded PVA: alginate fibrous scaffold.

Tough scaffolds comprised of aligned and conductive fibers are promising for peripheral nerve regeneration due to their unique mechanical and electrical properties. Several studies have confirmed that electrical stimulation can control the axonal extension in vitro. However, the stimulatory effects of scaffold architecture and electrical stimulation have not yet been investigated in detail. Here, we assessed a comparison between aligned and random fibers made of graphene (Gr) embedded sodium alginate (SA) polyvinyl alcohol (PVA) (Gr-AP scaffolds) for peripheral nerve engineering. The effects of applied electrical stimulation and orientation of the fabricated fibers on the in vitro attachment, alignment, and proliferation of PC12 cells (a rat neuronal cell line) were investigated. The results revealed that the aligned fibrous Gr-AP scaffolds closely mimicked the anisotropic structure of the native sciatic nerve. Aligned fibrous Gr-AP scaffolds significantly improved mechanical properties as well as cell-scaffold integration compared to random fibrous scaffolds. In addition, electrical stimulation significantly improved PC12 cell proliferation. In summary, our findings revealed that aligned fibrous Gr-AP scaffolds offered superior mechanical characteristics and structural properties that enhanced neural cell-substrate interactions, resulting in a promising construct for nerve tissue regeneration.

Nanohybrid hydrogels of laponite: PVA-Alginate as a potential wound healing material.

The aim of this study was to develop a novel nanohybrid interpenetrating network hydrogel composed of laponite:polyvinyl alcohol (PVA)-alginate (LAP:PVA-Alginate) with adjustable mechanical, physical and biological properties for wound healing application. Results demonstrated that compared to PVA-Alginate, mechanical strength of LAP:PVA-Alginate significantly enhanced (upon 2 times). Moreover, incorporation of 2wt.% laponite reduced swelling ability (3 times) and degradation ratio (1.2 times) originating from effective enhancement of crosslinking density in the nanohybrid hydrogels. Furthermore, nanohybrid hydrogels revealed admirable biocompatibility against MG63 and fibroblast cells. Noticeably, MTT assay demonstrated that fibroblast proliferation significantly enhanced on 0.5wt.% LAP:PVA-alginate compared to PVA-alginate. Moreover, hemolysis and clotting tests indicated that the nanohybrid hydrogels promoted hemostasis which could be helpful in the wound dressing. Therefore, the synergistic effects of the nanohybrid hydrogels such as superior mechanical properties, adjustable degradation rate and admirable biocompatibility and hemolysis make them a desirable candidate for wound healing process.

A facile one-step strategy for development of a double network fibrous scaffold for nerve tissue engineering.

The aim of this study was to develop a novel double network scaffold composed of polycaprolactone fumarate (PCLF) and eggshell membrane (ESM) (ESM:PCLF) by using the vacuum infiltration method. Compared to ESM, the mechanical properties of double network scaffold were significantly improved, depending on the solvents applied for double network scaffold formation; acetic acid and dichloromethane. Noticeably, the toughness and strength of double network scaffold prepared using acetic acid were significantly improved compared to ESM (26.6 and 25 times, respectively) attributed to the existence of hydrophilic functional groups in acetic acid which made ESM flexible to absorb further PCLF solution. To assess the effect of double network formation on the biological behavior of ESM, the attachment, proliferation and spreading of PC12 cells cultured on the ESM:PCLF scaffolds were evaluated. Results revealed that the number of cells attached on double network ESM:PCLF scaffold were nearly similar to ESM and significantly higher than that of on the tissue culture plate (2.6 times) and PCLF film (1.7 times). It is envisioned that the offered ESM:PCLF double network scaffold might have great potential to develop the constructs for nerve regeneration.