Ankle and Foot Arthroplasty and Prosthesis: A Review on the Current and Upcoming State of Designs and Manufacturing.

The foot and ankle serve vital roles in weight bearing, balance, and flexibility but are susceptible to many diverse ailments, making treatment difficult. More commonly, Total Ankle Arthroplasty (TAA) and Total Talus Replacement (TTR) are used for patients with ankle degeneration and avascular necrosis of the talus, respectively. Ankle prosthesis and orthosis are also indicated for use with lower limb extremity amputations or locomotor disability, leading to the development of powered exoskeletons. However, patient outcomes remain suboptimal, commonly due to the misfitting of implants to the patient-specific anatomy. Additive manufacturing (AM) is being used to create customized, patient-specific implants and porous implant cages that provide structural support while allowing for increased bony ingrowth and to develop customized, lightweight exoskeletons with multifunctional actuators. AM implants and devices have shown success in preserving stability and mobility of the joint and achieving fast recovery, as well as significant improvements in gait rehabilitation, gait assistance, and strength for patients. This review of the literature highlights various devices and technologies currently used for foot and ankle prosthesis and orthosis with deep insight into improvements from historical technologies, manufacturing methods, and future developments in the biomedical space.

The Inflammatory Contribution of B-Lymphocytes and Neutrophils in Progression to Osteoporosis.

Osteoporosis is a bone disease characterized by structural deterioration and low bone mass, leading to fractures and significant health complications. In this review, we summarize the mechanisms by which B-lymphocytes and neutrophils contribute to the development of osteoporosis and potential therapeutics targeting these immune mediators to reduce the proinflammatory milieu. B-lymphocytes-typically appreciated for their canonical role in adaptive, humoral immunity-have emerged as critical regulators of bone remodeling. B-lymphocytes communicate with osteoclasts and osteoblasts through various cytokines, including IL-7, RANK, and OPG. In inflammatory conditions, B-lymphocytes promote osteoclast activation and differentiation. However, B-lymphocytes also possess immunomodulatory properties, with regulatory B-lymphocytes (Bregs) secreting TGF-ß1 to restrain pathogenic osteoclastogenesis. Neutrophils, the body's most prevalent leukocyte, also contribute to the proinflammatory environment that leads to osteoporotic bone remodeling. In aged individuals, neutrophils display reduced chemotaxis, phagocytosis, and apoptosis. Understanding the delicate interplay between B-lymphocytes and neutrophils in the context of impaired bone metabolism is crucial for targeted therapies for osteoporosis.

Effects of Different Titanium Surfaces Created by 3D Printing Methods, Particle Sizes, and Acid Etching on Protein Adsorption and Cell Adhesion, Proliferation, and Differentiation.

The surfaces of 3D printed titanium prostheses have major impacts on the clinical performance of the prostheses. To investigate the surface effects of the products generated by 3D printed titanium on osseointegration, six surface types of titanium discs produced by the direct metal laser sintering (DMLS) and electron beam melting (EBM) methods, with two sizes of titanium particles and post-printing acid etching, were used to examine the surface topography and to explore the protein adsorption, pro-inflammatory cytokine gene expressions, and MC3T3-E1 cell adhesion, proliferation, and differentiation. The EBM-printed disc showed a stripy and smooth surface without evidence of the particles used, while the DMLS surface contained many particles. After acid etching, small particles on the DMLS surface were removed, whereas the large particles were left. Moreover, distinct proteins with low molecular weights were attached to the 3D printed titanium discs but not to the pre-printing titanium particles. The small titanium particles stimulated the highest TNF-α and IL-6 gene expressions at 24 h. The alizarin red content and osteocalcin gene expression at day 21 were the highest in the groups of acid-etched discs printed by DMLS with the small particles and by EBM. Therefore, the acid-treated surfaces without particles favor osteogenic differentiation. The surface design of 3D printed titanium prostheses should be based on their clinical applications.

The Involvement of Neutrophils in the Pathophysiology and Treatment of Osteoarthritis.

Osteoarthritis (OA) is a chronic disability that significantly impairs quality of life. OA is one of the most prevalent joint pathologies in the world, characterized by joint pain and stiffness due to the degeneration of articular cartilage and the remodeling of subchondral bone. OA pathogenesis is unique in that it involves simultaneous reparative and degradative mechanisms. Low-grade inflammation as opposed to high-grade allows for this coexistence. Previously, macrophages and T cells have been identified as playing major roles in the inflammation and destruction of OA joints, but recent studies have demonstrated that neutrophils also contribute to the pathogenesis. Neutrophils are the first immune cells to enter the synovium after joint injury, and neutrophilic activity is indispensably a requisite for the progression of OA. Neutrophils act through multiple mechanisms including tissue degeneration via neutrophil elastase (NE), osteophyte development, and the release of inflammatory cytokines and chemokines. As the actions of neutrophils in OA are discovered, the potential for novel therapeutic targets as well as diagnostic methods are revealed. The use of chondrogenic progenitor cells (CPCs), microRNAs, and exosomes are among the newest therapeutic advances in OA treatment, and this review reveals how they can be used to mitigate destructive neutrophil activity.

Rat Calvarial Bone Regeneration by 3D-Printed ß-Tricalcium Phosphate Incorporating MicroRNA-200c.

Advanced fabrication methods for bone grafts designed to match defect sites that combine biodegradable, osteoconductive materials with potent, osteoinductive biologics would significantly impact the clinical treatment of large bone defects. In this study, we engineered synthetic bone grafts using a hybrid approach that combined three-dimensional (3D-)printed biodegradable, osteoconductive ß-tricalcium phosphate (ß-TCP) with osteoinductive microRNA(miR)-200c. 3D-printed ß-TCP scaffolds were fabricated utilizing a suspension-enclosing projection-stereolithography (SEPS) process to produce constructs with reproducible microarchitectures that enhanced the osteoconductive properties of ß-TCP. Collagen coating on 3D-printed ß-TCP scaffolds slowed the release of plasmid DNA encoding miR-200c compared to noncoated constructs. 3D-printed ß-TCP scaffolds coated with miR-200c-incorporated collagen increased the transfection efficiency of miR-200c of both rat and human BMSCs and additionally increased osteogenic differentiation of hBMSCs in vitro. Furthermore, miR-200c-incorporated scaffolds significantly enhanced bone regeneration in critical-sized rat calvarial defects. These results strongly indicate that bone grafts combining SEPS 3D-printed osteoconductive biomaterial-based scaffolds with osteoinductive miR-200c can be used as superior bone substitutes for the clinical treatment of large bone defects.

Plasmid encoding microRNA-200c ameliorates periodontitis and systemic inflammation in obese mice.

The present study was conducted to characterize microRNA-200c (miR-200c) and its regulators in adipogenic differentiation, obesity, and periodontitis in obese subjects (PiOSs), and to determine the therapeutic efficacy of plasmid DNA encoding miR-200c as a treatment for PiOSs. We report that highly expressed miR-200c in gingival tissues was downregulated in diet-induced obese (DIO) mice and during adipogenic differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). Local injection of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) in the maxilla interdental gingiva of DIO mice reduced miR-200c in gingival and adipose tissues and induced periodontal inflammation associated with systemic elevation of interleukin-6 (IL-6) and impaired glucose tolerance. The inhibitory functions of Pg-LPS and IL-6 on miR-200c and their effectiveness on Zeb1 were confirmed in vitro. Injection of naked plasmid DNA encoding miR-200c into the gingiva effectively rescued miR-200c downregulation, prevented periodontal and systemic inflammation, and alleviated the impaired glucose metabolism in obese mice with LPS-induced periodontitis. Increased circulating exosomal miR-200c and its function on suppressing proinflammatory cytokines and adipogenesis explained the mechanism(s) of gingival application of miR-200c in attenuating systemic inflammation in PiOSs. These results demonstrated that miR-200c reduced by Pg-LPS and IL-6 in periodontitis and obesity might lead to the pathogenesis of PiOSs, and upregulation of miR-200c in the gingiva presents a therapeutic approach for PiOSs.

Enhancement of MicroRNA-200c on Osteogenic Differentiation and Bone Regeneration by Targeting Sox2-Mediated Wnt Signaling and Klf4.

MicroRNA (miR)-200c functions in antitumorigenesis and mediates inflammation and osteogenic differentiation. In this study, we discovered that miR-200c was upregulated in human bone marrow mesenchymal stromal cells (hBMSCs) during osteogenic differentiation. Inhibition of endogenous miR-200c resulted in downregulated osteogenic differentiation of hBMSCs and reduced bone volume in the maxilla and mandible of a transgenic mouse model. Overexpression of miR-200c by transfection of naked plasmid DNA (pDNA) encoding miR-200c significantly promoted the biomarkers of osteogenic differentiation in hBMSCs, including alkaline phosphatase, Runt-related transcription factor 2, osteocalcin, and mineral deposition. The pDNA encoding miR-200c also significantly enhanced bone formation and regeneration in calvarial defects of rat models. In addition, miR-200c overexpression was shown to downregulate SRY (sex determining region Y)-box 2 (Sox2) and Kruppel-like factor 4 by directly targeting 3'-untranslated regions and upregulate the activity of Wnt signaling inhibited by Sox2. These results strongly indicated that miR-200c may serve as a unique osteoinductive agent applied for bone healing and regeneration.

MicroRNA-200c Attenuates Periodontitis by Modulating Proinflammatory and Osteoclastogenic Mediators.

This study tested whether microRNA (miR)-200c can attenuate the inflammation and alveolar bone resorption in periodontitis by using an in vitro and a rat model. Polyethylenimine (PEI) was used to facilitate the transfection of plasmid DNA encoding miR-200c into primary human gingival fibroblasts (HGFs) and gingival tissues of rats. We first analyzed how proinflammatory and osteoclastogenic mediators in HGFs with overexpression of miR-200c responded to Porphyromonas gingivalis lipopolysaccharide (LPS-PG) challenge in vitro. We observed that overexpression of miR-200c significantly reduced interleukin (IL)-6 and 8 and repressed interferon-related developmental regulator-1 (IFRD1) in HGFs. miR-200c also downregulated p65 and p50. In a rat model of periodontitis induced by an LPS injection at the gingival sulcus of the second maxillary molar (M2), we analyzed how the mediators in rat gingiva and alveolar bone resorption responded to miR-200c treatment by a local injection of PEI-plasmid miR-200 nanoplexes. We observed that the local injection of miR-200c significantly upregulated miR-200c expression in gingiva and reduced IL-6, IL-8, IFRD1, and the ratio of receptor activator of nuclear factor kappa-B ligand/osteoprotegerin. Using micro-computed tomography analysis and histomorphometry, we further confirmed that local treatment with miR-200c effectively protected alveolar bone resorption in the rat model of periodontitis by reducing the distance between the cemento-enamel junction and the alveolar bone crest and the inter-radicular space in the upper maxilla at M2. These findings imply that miR-200c may serve as a unique means to prevent periodontitis and associated bone loss.

Strontium-releasing fluorapatite glass-ceramic scaffolds: Structural characterization and in vivo performance.

There is increasing interest in biodegradable ceramic scaffolds for bone tissue engineering capable of in situ delivery of ionic species favoring bone formation. Strontium has been shown to be osteogenic, but strontium-containing drugs such as strontium ranelate, used in Europe for the treatment of osteoporosis, are now restricted due to clinical evidence of systemic effects. By doping fluorapatite-based glasses with strontium, we developed ceramic scaffolds with fully interconnected macroporosity and cell size similar to that of cancellous bone, that are also capable of releasing strontium. The crystallization behavior, investigated by XRD and SEM, revealed the formation of akermanite and fluorapatite at the surface of strontium-free glass-ceramic scaffolds, and strontium-substituted fluorapatite at the surface of the strontium-doped scaffolds. At 8 weeks after implantation in a rat calvarial critical size defect, scaffolds doped with the highest amount of strontium led to the highest mineral apposition rate. A significantly higher amount of newly-formed bone was found with the strontium-free glass-ceramic scaffold, and possibly linked to the presence of akermanite at the scaffold surface. We demonstrate by energy dispersive XRF analyses of skull sections that strontium was present in newly formed bone with the strontium-doped scaffolds, while a significant amount of fluorine was incorporated in newly formed bone, regardless of composition or crystallization state. STATEMENT OF SIGNIFICANCE: The present work demonstrates the in vivo action of strontium-containing glass-ceramic scaffolds. These bone graft substitutes are targeted at non load-bearing bone defects. Results show that strontium is successfully incorporated in newly formed bone. This is associated with a significantly higher Mineral Apposition Rate. The benefits of in situ release of strontium are demonstrated. The broader scientific impact of this works builds on the concept of resorbable ceramic scaffolds as reservoirs of ionic species capable of enhancing bone regeneration.

Controlled and Sequential Delivery of Fluorophores from 3D Printed Alginate-PLGA Tubes.

Controlled drug delivery systems, that include sequential and/or sustained drug delivery, have been utilized to enhance the therapeutic effects of many current drugs by effectively delivering drugs in a time-dependent and repeatable manner. In this study, with the aid of 3D printing technology, a novel drug delivery device was fabricated and tested to evaluate sequential delivery functionality. With an alginate shell and a poly(lactic-co-glycolic acid) (PLGA) core, the fabricated tubes displayed sequential release of distinct fluorescent dyes and showed no cytotoxicity when incubated with the human embryonic kidney (HEK293) cell line or bone marrow stromal stem cells (BMSC). The controlled differential release of drugs or proteins through such a delivery system has the potential to be used in a wide variety of biomedical applications from treating cancer to regenerative medicine.

Correction: MicroRNA-200c Represses IL-6, IL-8, and CCL-5 Expression and Enhances Osteogenic Differentiation.

[This corrects the article DOI: 10.1371/journal.pone.0160915.].

MicroRNA-200c Represses IL-6, IL-8, and CCL-5 Expression and Enhances Osteogenic Differentiation.

MicroRNAs (miRs) regulate inflammation and BMP antagonists, thus they have potential uses as therapeutic reagents. However, the molecular function of miR-200c in modulating proinflammatory and bone metabolic mediators and osteogenic differentiation is not known. After miR-200c was transduced into a human embryonic palatal mesenchyme (HEPM) (a cell line of preosteoblasts), using lentiviral vectors, the resulting miR-200c overexpression increased osteogenic differentiation biomarkers, including osteocalcin (OCN) transcripts and calcium content. miR-200c expression also down-regulated interleukin (IL)-6, IL-8, and chemokine (C-C motif) ligand (CCL)-5 under lipopolysaccharide (LPS) stimulation and increased osteoprotegerin (OPG) in these cells. miR-200c directly regulates the expression of IL-6, IL-8 and CCL-5 transcripts by binding to their 3'UTRs. A plasmid-based miR-200c inhibitor effectively reduces their binding activities. Additionally, miR-200c delivered using polyethylenimine (PEI) nanoparticles effectively inhibits IL-6, IL-8 and CCL-5 in primary human periodontal ligament fibroblasts and increases the biomarkers of osteogenic differentiation in human bone marrow mesenchymal stem cells (MSCs), including calcium content, ALP, and Runx2. These data demonstrate that miR-200c represses IL-6, IL-8 and CCL-5 and improves osteogenic differentiation. miR-200c may potentially be used as an effective means to prevent periodontitis-associated bone loss by arresting inflammation and osteoclastogenesis and enhancing bone regeneration.

Microfabrication of scaffold-free tissue strands for three-dimensional tissue engineering.

In this note, we report a practical and efficient method based on a coaxial extrusion and microinjection technique for biofabrication of scaffold-free tissue strands. Tissue strands were obtained using tubular alginate conduits as mini-capsules with well-defined permeability and mechanical properties, where their removal by ionic decrosslinking allowed the formation of scaffold-free cell aggregates in the form of cylindrical strands with well-defined morphology and geometry. Rat dermal fibroblasts and mouse insulinoma beta TC3 cells were used to fabricate both single-cellular and heterocellular tissue strands with high cell viability, self-assembling capability and the ability to express cell-specific functional markers. By taking advantage of tissue self-assembly, we succeeded in guiding the fusion of tissue strands to fabricate larger tissue patches. The presented approach enables fabrication of cell aggregates with controlled dimensions allowing highly long strands, which can be used for various applications, including fabrication of scale-up complex tissues and of tissue models for drug screening and cancer studies.

In Vitro Study of Directly Bioprinted Perfusable Vasculature Conduits.

The ability to create three dimensional (3D) thick tissues is still a major tissue engineering challenge. It requires the development of a suitable vascular supply for an efficient media exchange. An integrated vasculature network is particularly needed when building thick functional tissues and/or organs with high metabolic activities, such as the heart, liver and pancreas. In this work, human umbilical vein smooth muscle cells (HUVSMCs) were encapsulated in sodium alginate and printed in the form of vasculature conduits using a coaxial deposition system. Detailed investigations were performed to understand the dehydration, swelling and degradation characteristics of printed conduits. In addition, because perfusional, permeable and mechanical properties are unique characteristics of natural blood vessels, for printed conduits these properties were also explored in this work. The results show that cells encapsulated in conduits had good proliferation activities and that their viability increased during prolonged in vitro culture. Deposition of smooth muscle matrix and collagen was observed around the peripheral and luminal surface in long-term cultured cellular vascular conduit through histology studies.

Engineering bone tissue using human dental pulp stem cells and an osteogenic collagen-hydroxyapatite-poly (L-lactide-co-ε-caprolactone) scaffold.

The aim of this study was to design a new natural/synthetic bioactive bone scaffold for potential use in bone replacement applications. We developed a tri-component osteogenic composite scaffold made of collagen (Coll), hydroxyapatite (HA) and poly(l-lactide-co-ε-caprolactone) (PLCL). This Coll/HA/PLCL composite scaffold was combined with human osteoblast-like cells obtained by differentiation of dental pulp stem cells (DPSCs) to engineer bone tissue in vitro. Results show that the 3D Coll/HA/PLCL composite scaffold was highly porous, thereby enabling osteoblast-like cell adhesion and growth. Cultured in the Coll/HA/PLCL scaffold, the osteoblast-like cells expressed different osteogenic genes, produced alkaline phosphatase and formed nodules more than did PLCL alone. Micro-CT analyses revealed a significant (30%) increase of tissue mineralisation on the surface as well as inside of the Coll/HA/PLCL scaffold, thus confirming its effectiveness as a bone regeneration platform.

A novel collagen/hydroxyapatite/poly(lactide-co-ε-caprolactone) biodegradable and bioactive 3D porous scaffold for bone regeneration.

The goal of this study was to design a nontoxic scaffold with both composition and microstructure suitable for bone engineering using collagen (Coll), hydroxyapatite (HA), and poly(lactide-co-ε-caprolactone) (PLCL). Mineralized type I Coll was produced by direct nucleation of HA particles inside self-assembled Coll fibers to obtain a Coll/HA complex, which was then added to dissolved PLCL (70:30) in 1,4-dioxane. A 3D porous Coll/HA/PLCL scaffold was subsequently produced through freeze-drying/lyophilization and salt-leaching procedures. The resulting Coll/HA/PLCL scaffold displayed a high uniform porosity and highly interconnected pores. X-ray photoelectron spectrometer and Fourier transform infrared analyses revealed the presence of both collagen and HA particles on the surface of the Coll/HA/PLCL scaffold. Proliferation assay, microscopic observations, and gene analysis with quantitative RT-PCR showed that osteoblast cells were able to attach, proliferate, and maintain an osteoblastlike phenotype when cultured on the Coll/HA/PLCL scaffold. In summary, we produced a nontoxic scaffold that contains natural polymers (Coll and HA) and synthetic polymer (PLCL). Through its chemical composition and porous morphology, this scaffold may be useful for osteoblast growth, differentiation, and bone tissue formation.

Bioactivating electrically conducting polypyrrole with fibronectin and bovine serum albumin.

Electrically conducting polypyrrole (PPy) and its composite materials are useful in interfacing electrical components and cells or living tissue. In recent years, significant efforts have been made to bioactivate PPy by incorporating biomolecules. The main objective of this work was to chemically bioactivate PPy particules by incorporating fibronectin (FN) and bovine serum albumin (BSA). Modified PPy particles were synthesized through a water-in-oil emulsion polymerization. XPS and FTIR confirmed the presence of biomolecules on the PPy particles, and the surface morphology was observed by SEM. A four-point probe was used to measure the conductivity of the newly synthesized PPy particles, which was in the range of 10(-1) S cm(-1). Conductive biodegradable membranes were prepared with 5 and 10% (wt/wt) PPy to poly(L,L-lactide) (PPy/PLLA). The contact angles of each synthesized membrane were approximately 75 degrees , supporting their usefulness for cell culture. The cultured human skin fibroblasts demonstrated normal morphology and significantly higher adhesion and spreading on the PPy/PLLA approximately FN membrane than on the unmodified PPy/PLLA membrane. On the other hand, the PPy/PLLA approximately BSA membranes showed decreased cell adhesion. Bioactivated PPy may be useful in tissue engineering to fabricate conducting biodegradable scaffolds with either improved or reduced cell adhesion properties for various cell culture and in vivo applications.