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Ample proof showed that non-coding RNAs (ncRNAs) play a crucial role in proliferation and differentiation of osteoblasts and bone marrow stromal cells (BMSCs). Varied forms of biophysical stimuli like mechanical strain, fluid shear stress (FSS), microgravity and vibration are verified to regulate ncRNAs expression in osteogenic differentiation and influence the expression of target genes associated with osteogenic differentiation and ultimately regulate bone formation. The consequences of biophysical stimulation on osteogenic differentiation validate the prospect of exercise for the prevention and treatment of osteoporosis. In this review, we tend to summarize the studies on regulation of osteogenic differentiation by ncRNAs beneath biophysical stimulation and facilitate to reveal the regulatory mechanism of biophysical stimulation on ncRNAs, and provide an update for the prevention of bone metabolism diseases by exercise.

Objectives: The purpose of this work was to review the recent advances in biophysical stimulation of MSC for bone regeneration with particular relevance in the tissue engineering field. Methods/Statistical Analysis: The review process had three steps: First, by the use of databases available, the principal findings published related to the different types of biophysical stimulation applied to Mesenchymal Stem cells (MSC) for bone tissue regeneration were compiled. Second, the principal characteristics such as historical relevance, conditions of operation, signaling, and principal results were obtained from each study. And third, considering the above characteristics, a description of each study was realized. Findings: This review highlighted the following findings: a) The capacity of MSC for differentiating to multiple lineages have attracted attention in regenerative medicine applications; b) Biophysical stimulation is an alternative in order to promote the osteodifferentiation of MSC; c) During the process of application of this type of stimulation, the generation of biochemical signals which is related to the changes in the environment of the cell (i.e., cell attachment, proliferation, and differentiation) are generated; and d) Despite a large number of studies published in this area, these do not explain clearly the mechanisms related to the generation of these signaling produced by the biophysical effects (i.e., mechanical, electrical, and electromagnetic). Furthermore, in this review, a compilation of the last five years was done, which emphasize in the aspect historical, conditions of operation, and biochemical signaling generated of each type of biophysical stimulation of MSC for osteodifferentiation. Application/ Improvements: Biophysical stimulation causes multiple effects on the cell environment, producing changes in its morphology, proliferation, and differentiation. The above is important in the biophysical stimulation of MSC for bone regeneration. Keywords: Biophysical Stimulation, Bone Regeneration, Osteodifferentiation, Stem Cells, Tissue Engineering

Muscle atrophy is defined as the decrease in the size and number of muscle fibers, and is associated with injury to muscle structures. Recently, biophysical therapies using laser, ultrasound, and vibration has been widely used to improve muscle atrophy. However, although the effects of these stimuli seem to be similar, the mechanisms by which they stimulate biological tissue may be different. From this point of view, we expected that it would be possible to produce synergetic effects through combining these three different types of biophysical stimuli on biological tissues, based on the therapeutic benefit of each stimulus. For this, 35 males, 12-week old, C57BL/6 mice (21 ± 1.2 g), were randomly assigned to five groups: a) a sciatic nerve neurectomized “control” group (C, n = 7), b) a MILNS (Minimally Invasive Laser Needle System) therapy after sciatic nerve neurectomized group (L, n = 7), c) a LIPUS (Low-Intensity Pulsed Ultrasound) therapy after sciatic nerve neurectomized group (U, n = 7), e) a PVS (Partial Vibration Stimulation) therapy after sciatic nerve neurectomized group (V, n = 7), and e) a multimodal biophysical stimulation after sciatic nerve neurectomized group (MS, n = 7).

Stem cell therapy shows great potential in the treatment of neurodegenerative diseases, in which efficient neural differentiation of stem cells is still challenging. In this work, a new strategy based on magnetic iron oxide nanoparticles (MIONs) was developed to induce neural differentiation of mouse embryonic stem cells (mESCs). The mechanical forces of the magnetic iron oxide nanoparticles and the magnetic field significantly promoted the differentiation of the embryonic stem cells into nerve cells, and the promotion effect did not depend on the neural differentiation medium. Most importantly, under a combination of the three factors, neural differentiation of mESCs was significantly promoted, and the expression level of mature nerve cell marker genes was increased by 60 times. The results indicated that the synergistic effect of magnetic iron oxide nanoparticles, a magnetic field and a neural differentiation medium could significantly promote the neural differentiation of embryonic stem cells. What's more, our study provided a harmless, permeating, remote controllable, and biophysical method for promoting the neural differentiation of stem cells, which had great potential for practical applications.

A plethora of antimicrobial strategies are being developed to address prosthetic infection. The currently available methods for implant infection treatment include the use of antibiotics and revision surgery. Among the bacterial strains, Staphylococcus species pose significant challenges particularly, with regard to hospital acquired infections. In order to combat such life threatening infectious diseases, researchers have developed implantable biomaterials incorporating nanoparticles, antimicrobial reinforcements, surface coatings, slippery/non-adhesive and contact killing surfaces. This review discusses a few of the biomaterial and biophysical antimicrobial strategies, which are in the developmental stage and actively being pursued by several research groups. The clinical efficacy of biophysical stimulation methods such as ultrasound, electric and magnetic field treatments against prosthetic infection depends critically on the stimulation protocol and parameters of the treatment modality. A common thread among the three biophysical stimulation methods is the mechanism of bactericidal action, which is centered on biophysical rupture of bacterial membranes, the generation of reactive oxygen species (ROS) and bacterial membrane depolarization evoked by the interference of essential ion-transport. Although the extent of antimicrobial effect, normally achieved through biophysical stimulation protocol is insufficient to warrant therapeutic application, a combination of antibiotic/ROS inducing agents and biophysical stimulation methods can elicit a clinically relevant reduction in viable bacterial numbers. In this review, we present a detailed account of both the biomaterial and biophysical approaches for achieving maximum bacterial inactivation. Summarizing, the biophysical stimulation methods in a combinatorial manner with material based strategies can be a more potent solution to control bacterial infections. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2174-2190, 2017.

Purpose: The purpose of the present study was to evaluate the effects of pulsed electromagnetic fields (PEMFs) on clinical outcome in patients who underwent arthroscopic matrix-assisted autologous chondrocyte implantation (MACI) for chondral lesions of the knee. Methods: Thirty patients affected by grade III and IV International Cartilage Repair Society chondral lesions of the knee underwent MACI. After surgery, patients were randomly assigned to either experimental group (PEMFs 4 h per day for 60 days) or control group. Clinical outcome was evaluated through International Knee Documentation Committee (IKDC) subjective knee evaluation form, Visual Analog Scale, Short Form-36 (SF-36) and EuroQoL before surgery and 1, 2, 6, and 60 months postoperative. Results: Mean size of chondral lesion was 2.4 ± 0.6 cm2in the PEMFs group and 2.5 ± 0.5 cm2in the control one. No differences were found between groups at baseline. IKDC score increased in both groups till 6 months, but afterward improvement was observed only in the experimental group with a significant difference between groups at 60 months (p = 0.001). A significant difference between groups was recorded at 60 months for SF-36 (p = 0.006) and EuroQol (p = 0.020). A significant pain reduction was observed in the experimental group at 1-, 2- and 60-month follow-up. Conclusion: Biophysical stimulation with PEMFs improves clinical outcome after arthroscopic MACI for chondral lesions of the knee in the short- and long-term follow-up. Biophysical stimulation should be considered as an effective tool in order to ameliorate clinical results of regenerative medicine. The use of PEMFs represents an innovative therapeutic approach for the survival of cartilage-engineered constructs and consequently the success of orthopaedic surgery. Level of evidence: II.

Stem cells proliferate by undergoing self-renewal and differentiate into multiple cell lineages in response to biochemical and biophysical stimuli. Various biochemical cues such as growth factors, nucleic acids, chemical reagents, and small molecules have been used to induce stem cell differentiation or reprogramming or to maintain their pluripotency. Moreover, biophysical cues such as matrix stiffness, substrate topography, and external stress and strain play a major role in modulating stem cell behavior. In this chapter, we have summarized microenvironmental regulation of stem cell behavior through biochemical and biophysical stimulation.

Tissue engineering is a promising therapeutic strategy to regenerate skeletal muscle. However, ex vivo cultivation methods typically result in a low differentiation efficiency of stem cells as well as grafts that resemble the native tissues morphologically, but lack contractile function. The application of biomimetic tensile strain provides a potent stimulus for enhancing myogenic differentiation and engineering functional skeletal muscle grafts. We reviewed integrin-dependent mechanisms that potentially link mechanotransduction pathways to the upregulation of myogenic genes. Yet, gaps in our understanding make it challenging to use these pathways to theoretically determine optimal ex vivo strain regimens. A multitude of strain protocols have been applied to in vitro cultures for the cultivation of myogenic progenitors (adipose- and bone marrow-derived stem cells and satellite cells) and transformed murine myoblasts, C2C12s. Strain regimens are characterized by orientation, amplitude, and time-dependent factors (effective frequency, duration, and the rest period between successive strain cycles). Analysis of published data has identified possible minimum/maximum values for these parameters and suggests that uniaxial strains may be more potent than biaxial strains, possibly because they more closely mimic physiologic strain profiles. The application of these biophysical stimuli for engineering 3D skeletal muscle grafts is nontrivial and typically requires custom-designed bioreactors used in combination with biomaterial scaffolds. Consideration of the physical properties of these scaffolds is critical for effective transmission of the applied strains to encapsulated cells. Taken together, these studies demonstrate that biomimetic tensile strain generally results in improved myogenic outcomes in myogenic progenitors and differentiated myoblasts. However, for 3D systems, the optimization of the strain regimen may require the entire system including cells, biomaterials, and bioreactor, to be considered in tandem.

Author(s): Lin, Michael Y; Frieboes, Laura S; Forootan, Maryam; Palispis, Winnie A; Mozaffar, Tahseen; Jafari, Matiar; Steward, Oswald; Gall, Christine M; Gupta, Ranjan | Abstract: ObjectiveChronic nerve compression (CNC) injuries occur when peripheral nerves are subjected to sustained mechanical forces, with increasing evidence implicating Schwann cells as key mediators. Integrins, a family of transmembrane adhesion molecules that are capable of intracellular signaling, have been implicated in a variety of biological processes such as myelination and nerve regeneration. In this study, we seek to define the physical stimuli mediating demyelination and to determine whether integrin plays a role in the demyelinating response.MethodsWe used a previously described in vitro model of CNC injury where myelinating neuron-Schwann cell cocultures were subjected to independent manipulations of hydrostatic pressure, hypoxia, and glucose deprivation in a custom bioreactor. We assessed whether demyelination increased in response to applied manipulation and determined whether integrin-associated signaling cascades are upregulated.ResultsBiophysical stimulation of neural tissue induced demyelination and Schwann cell proliferation without neuronal or glial cytotoxicity or apoptosis. Although glucose deprivation and hypoxia independently had minor effects on myelin stability, together they potentiated the demyelinating effects of hydrostatic compression, and in combination, significantly destabilized myelin. Biophysical stimuli transiently increased phosphorylation of the integrin-associated tyrosine kinase Src within Schwann cells. Silencing this integrin signaling cascade blocked Src activation and prevented pressure-induced demyelination. Colocalization analysis indicated that Src is localized within Schwann cells.InterpretationThese results indicate that myelin is sensitive to CNC injury and support the novel concept that myelinating cocultures respond directly to mechanical loading via activating an integrin signaling cascade.

Background: Musculoskeletal disorders are regarded as a major cause of worldwide morbidity and disability, and they result in huge costs for national health care systems. Traditional therapies frequently turned out to be poorly effective in treating bone, cartilage, and tendon disorders or joint degeneration. As a consequence, the development of novel biological therapies that can treat more effectively these conditions should be the highest priority in regenerative medicine. Main body of the abstract: Mesenchymal stem cells (MSCs) represent one of the most promising tools in musculoskeletal tissue regenerative medicine, thanks to their proliferation and differentiation potential and their immunomodulatory and trophic ability. Indeed, MSC-based approaches have been proposed for the treatment of almost all orthopedic conditions, starting from different cell sources, alone or in combination with scaffolds and growth factors, and in one-step or two-step procedures. While all these approaches would require cell harvesting and transplantation, the possibility to stimulate the endogenous MSCs to enhance their tissue homeostasis activity represents a less-invasive and cost-effective therapeutic strategy. Nowadays, the role of tissue-specific resident stem cells as possible therapeutic target in degenerative pathologies is underinvestigated. Biophysical stimulations, and in particular extracorporeal shock waves treatment and pulsed electromagnetic fields, are able to induce proliferation and support differentiation of MSCs from different origins and affect their paracrine production of growth factors and cytokines. Short conclusions: The present review reports the attempts to exploit the resident stem cell potential in musculoskeletal pathologies, highlighting the role of MSCs as therapeutic target of currently applied biophysical treatments.

Biophysical stimulation to enhance bone fracture repair and bone regenerate maturation to restore its structural strength must rely on both the biological and biomechanical principle according to the local tissue environment and the type of mechanical stress to be born by the skeletal joint system. This paper reviews the possible interactions between biophysical stimuli and cellular responses in healing bone fractures and proceeds to speculate the prospects and limitations of different experimental models in evaluating and optimising such non-invasive interventions. It is important to realize that bone fracture repair has several pathways with various combinations of bone formation mechanisms, but there may only be one bone remodeling principle regulated by the hypothesis proposed by Wolff. There are different mechanical and biophysical stimuli that could provide effective augmentation of fracture healing and bone regenerate maturation. The key requirements of establishing these positive interactions are to define the precise cellular response to the stimulation signal in an in vitro environment and to use well-established animal models to quantify and optimise the therapeutic regimen in a time-dependent manner. This can only be achieved through research collaboration among different disciplines using scientific methodologies. In addition, the specific forms of biophysical stimulation and its dose effect and application timing must be carefully determined and validated. Technological advances in achieving focalized stimulus delivery with adjustable signal type and intensity, in the ability to monitor healing callus mechanical property non-invasively, and in the establishment of a robust knowledge base to develop effective and reliable treatment protocols are the essential pre-requisites to make biophysical stimulation acceptable in the main arena of health care. Finally, it is important to bear in mind that successful fracture repair or bone regeneration through callus distraction without adequate remodeling process through physiological loading would seriously undermine the value of biophysical stimulation in meeting the biomechanical demand of a long bone.

The effect of pulsed electromagnetic fields (PEMFs) on the integration of osteochondral autografts was evaluated in sheep. After osteochondral grafts were performed, the animals were treated with PEMFs for 6 h/day or sham-treated. Six animals were sacrificed at 1 month. Fourteen animals were treated for 2 months and sacrificed at 6 months. At 1 month, the osteogenic activity at the transplant-host subchondral bone interface was increased in PEMF-treated animals compared to controls. Articular cartilage was healthy in controls and stimulated animals. At 6 months, complete resorption was observed in four control grafts only. Cyst-like resorption areas were more frequent within the graft of sham-treated animals versus PEMF-treated. The average volume of the cysts was not significantly different between the two groups; nevertheless, analysis of the variance of the volumes demonstrated a significant difference. The histological score showed no significant differences between controls and stimulated animals, but the percentage of surface covered by fibrous tissue was higher in the control group than in the stimulated one. Interleukin-1 and tumor necrosis factor-alpha concentration in the synovial fluid was significantly lower, and transforming growth factor-beta1 was significantly higher, in PEMF-treated animals compared to controls. One month after osteochondral graft implantation, we observed larger bone formation in PEMF-treated grafts which favors early graft stabilization. In the long term, PEMF exposure limited the bone resorption in subchondral bone; furthermore, the cytokine profile in the synovial fluid was indicative of a more favorable articular environment for the graft.

Previous research on vertebrate limb regeneration indicates there are several mediating factors involved during the re-growth process. These factors are both biochemical and biophysical. While the phenomenon of adult limb regeneration does not occur naturally in mammalian species, prior research has focused mainly on biochemical modes of stimulating tissue growth and regeneration. The BioDome was aimed at developing a new experimental tool to permit the more systematic study of the impact of biophysical and biochemical factors on mammalian tissue regeneration. The BioDome is a multi-component sleeve assembly that encompasses the wound site of an amputated murine digit and provides an environment conducive to tissue regeneration. The studies showed that the BioDome was effective in supporting early stages of murine digit tip regeneration when combined with a porcine urinary bladder matrix (UBM) pepsin digest and electrical stimulation. The hydrated inner environment of the BioDome influenced regeneration, with additional effects seen with the application of electrical stimulation and pharmacological treatments.

Osteonecrosis of the femoral head is the endpoint of a disease process that results from insufficient blood flow and bone-tissue necrosis, leading to joint instability, collapse of the femoral head, arthritis of the joint, and total hip replacement. Pain is the most frequent clinical symptom. Both bone tissue and cartilage suffer when osteonecrosis of the femoral head develops. Stimulation with pulsed electromagnetic fields (PEMFs) has been shown to be useful for enhancing bone repair and for exerting a chondroprotective effect on articular cartilage. Two Italian studies on the treatment of avascular necrosis of the femoral head with PEMFs were presented in this review. In the first study, 68 patients suffering from avascular necrosis of the femoral head were treated with PEMFs in combination with core decompression and autologous bone grafts. The second one is a retrospective analysis of the results of treatment with PEMFs of 76 hips in 66 patients with osteonecrosis of the femoral head. In both studies clinical information and diagnostic imaging were collected at the beginning of the treatment and at the time of follow up. Statistical analysis was performed using chi-square test. Both authors hypothesize that the short-term effect of PEMF stimulation may be to protect the articular cartilage from the catabolic effect of inflammation and subchondral bone-marrow edema. The long-term effect of PEMF stimulation may be to promote osteogenic activity at the necrotic area and prevent trabecular fracture and subchondral bone collapse. PEMF stimulation represents an important therapeutic opportunity to resolve the Ficat stage-I or II disease or at least to delay the time until joint replacement becomes necessary.

The development of biophysical technologies for use in orthopedics is based on the discovery of the electrical properties of bone tissue in the 1950s and 1960s. The landmark study, first reported in 1954, on bone piezoelectric properties was conducted in Japan by Fukada and Yasuda (1). These authors measured an electric potential on deformation of dry bone. This work stimulated many research groups to investigate these findings further. By the early 1960s, several groups, notably those led by Bassett at Columbia University and Brighton at the University of Pennsylvania, reported the generation of electrical potentials in wet bone on mechanical deformation (2, 3, 4, 5). Similar observations were subsequently made in other tissues including collagen and cartilaginous tissues under mechanical stress (6, 7, 8).

Avascular necrosis (AVN) of the femur head represents a pathological condition of clinical-epidemiological importance. The uncertainties persisting with regard to the etiology, pathogenesis, and therapy justify the growing interest in this disease and necessitate the introduction of suitable protocols for diagnosis and therapy [1,2].

Endochondral ossification can be modulated by a number of biochemical and biophysical stimuli. This study uses the experimental model of decalcified bone matrix-induced endochondral ossification to examine the effect of one biophysical stimulus, an electromagnetic field, on chondrogenesis, calcification, and osteogenesis. A temporal acceleration and quantitative increase in sulfate incorporation, glycosaminoglycan content, and calcification suggests that the stimulation of endochondral ossification is due to an increase in extracellular matrix synthesis. The locus of that stimulation is identified in the mesenchymal stage of endochondral bone development, and stimulation at this stage is essential for accelerated bone formation. The data suggest that enhanced differentiation of mesenchymal stem cells present at this stage is most likely responsible for the increase in extracellular matrix synthesis and bone maturation.

Cells and tissues respond to a large variety of extracellular signals, including electromagnetic fields (EMF). Recent studies have demonstrated that combined AC and DC magnetic fields may couple specifically to ion dependent cellular processes. This coupling suggests an extraordinary potential for use of these combined magnetic fields for tissue healing applications in clinical situations. To this end, we have perfomed studies on in vitro osteoblast and in vivo rat osteoporosis model systems. Since osteoporosis is a result of impaired bone formation/bone resoprtion, we proposed to test whether direct osteoblast activation via calcium-dependent pathways would prevent bone loss in a model of hormonally induced osteoporosis. The cellular studies addressed the question of whether combined magnetic fields could induce cell proliferation, and whether this effect was based on autocrine growth factor (insulin-like growth factor; IGF-II) stimulation by osteoblasts.

Pulsed Electromagnetic Field (PEMF) is a non invasive, therapeutic form of low field magnetic stimulation that has been used for several years to supplement bone healing. It is known to generate pulsating magnetic frequencies within the body that accelerate the process of healing and reduce postoperative pain. The survival rate of dental implants over a 10-year observation has been reported to be higher than 90%. Success of dental implant therapy depends on the quality and quantity of available bone in which they are inserted. Implants with poor early or primary stability frequently may require additional time for osseointegration or may sometimes fail. Development of procedures which accelerate osseointegration of dental implants, reduce the period of healing, and lead to an early rehabilitation of the patient are required for successful oral rehabilitation. The potential for bone repair can be stimulated through non invasive adjunctive treatments such as application of pulsating electromagnetic field therapy, LowIntensity Pulsed Ultrasound (LIPUS), and Low-Level Laser Therapy (LLLT). These methods of biophysical stimulation of bone union were developed initially to enhance the healing of fractures, healing of bony non unions and have been hypothesised to improve implant osseointegration. This study sought to report latest trends in PEMF Therapy stimulations in oral tissues and its use to enhance the bone repair and regeneration. Pulsed electromagnetic field stimulation to induce bone regeneration mandates a broad range of settings that include magnetic field intensity, frequency, type of signals and duration of application etc. The present study analyses these clinical settings in published human trials and is expected to serve as a treatment guide for the clinicians to bring into their clinical use these strategies to improve bone regeneration and implant osseointegration in deficient and osteoporotic bone.

Compared to two-dimensional cell cultures, three-dimensional ones potentially allow recreating natural tissue environments with higher accuracy. The three-dimensional approach is being investigated in the field of tissue engineering targeting the reconstruction of various tissues, among which skeletal muscle. Skeletal muscle is an electroactive tissue which strongly relies upon interactions with the extracellular matrix for internal organization and mechanical function. Studying the optimization of myogenesis in vitro implies focusing on appropriate biomimetic stimuli, as biochemical and electrical ones. Here we present a three-dimensional polyurethane-based soft porous scaffold (porosity ~ 86%) with a Young's modulus in wet conditions close to the one of natural skeletal muscle tissue (~ 9 kPa). To study the effect of external stimuli on muscle cells, we functionalized the scaffold with extracellular matrix components (laminin and fibronectin) and observed an increase in myoblast proliferation over three days. Furthermore, the combination between laminin coating and electrical stimulation resulted in more spread and thicker myotubes compared to non-stimulated samples and samples receiving the single (non-combined) inputs. These results pave the way to the development of mature muscle tissue within three-dimensional soft scaffolds, through the combination of biochemical and electrical stimuli.

Pulsed electromagnetic fields (PEMFs) have been used to treat bone diseases, particularly nonunion healing. Although it is known that PEMFs promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs), to date PEMF molecular mechanisms remain not clearly elucidated. The Notch signalling is a highly conserved pathway that regulates cell fate decisions and skeletal development. The aim of this study was to investigate if the known PEMF-induced osteogenic effects may involve the modulation of the Notch pathway. To this purpose, during in vitro osteogenic differentiation of bone marrow hMSCs in the absence and in the presence of PEMFs, osteogenic markers (alkaline phosphatase activity, osteocalcin and matrix mineralization), the messenger ribonucleic acid expression of osteogenic transcription factors (Runx2, Dlx5, Osterix) as well as of Notch receptors (Notch1-4), their ligands (Jagged1, Dll1 and Dll4) and nuclear target genes (Hes1, Hes5, Hey1, Hey2) were investigated. PEMFs stimulated all osteogenic markers and increased the expression of Notch4, Dll4, Hey1, Hes1 and Hes5 in osteogenic medium compared to control. In the presence of DAPT and SAHM1, used as Notch pathway inhibitors, the expression of the osteogenic markers, including Runx2, Dlx5, Osterix, as well as Hes1 and Hes5 were significantly inhibited, both in unexposed and PEMF-exposed hMSCs. These results suggest that activation of Notch pathway is required for PEMFs-stimulated osteogenic differentiation. These new findings may be useful to improve autologous cell-based regeneration of bone defects in orthopaedics.

Pretreatment using various types of biophysical stimuli could provide appropriate potential to cells during construction of the engineered tissue in vitro. We hypothesized that multiple combinations of these biophysical stimuli could enhance osteogenic differentiation in vitro and bone formation in vivo. Cyclic strain, an electromagnetic field, and ultrasound were selected and combined as effective stimuli for osteogenic differentiation using a developed bioreactor. Here we report the experimental evaluation of the osteogenic effects of various combinations of three different biophysical stimuli in vitro and in vivo using human adipose-derived stem cells (ASCs). Osteogenic differentiation of ASCs was accelerated by multiple-combination biophysical stimulation in vitro. However, both single stimulation and double-combination stimulation were sufficient to accelerate bone regeneration in vivo, while the osteogenic marker expression of those groups was not as high as that of triple-combination stimulation in vitro. We inferred from these data that ASCs appropriately differentiated into the osteogenic lineage by biophysical stimulation could be a better option for accelerating bone formation in vivo than relatively undifferentiated or completely differentiated ASCs. Although many questions remain about the mechanisms of combined effects of various biophysical stimuli, this approach could be a more powerful tool for bone tissue regeneration.

Bioreactors that subject cell seeded scaffolds or hydrogels to biophysical stimulation have been used to improve the functionality of tissue engineered cartilage and to explore how such constructs might respond to the application of joint specific mechanical loading. Whether a particular cell type responds appropriately to physiological levels of biophysical stimulation could be considered a key determinant of its suitability for cartilage tissue engineering applications. The objective of this study was to determine the effects of dynamic compression on chondrogenesis of stem cells isolated from different tissue sources. Porcine bone marrow (BM) and infrapatellar fat pad (FP) derived stem cells were encapsulated in agarose hydrogels and cultured in a chondrogenic medium in free swelling (FS) conditions for 21 d, after which samples were subjected to dynamic compression (DC) of 10% strain (1 Hz, 1 h d(-1)) for a further 21 d. Both BM derived stem cells (BMSCs) and FP derived stem cells (FPSCs) were capable of generating cartilaginous tissues with near native levels of sulfated glycosaminoglycan (sGAG) content, although the spatial development of the engineered grafts strongly depended on the stem cell source. The mechanical properties of cartilage grafts generated from both stem cell sources also approached that observed in skeletally immature animals. Depending on the stem cell source and the donor, the application of DC either enhanced or had no significant effect on the functional development of cartilaginous grafts engineered using either BMSCs or FPSCs. BMSC seeded constructs subjected to DC stained less intensely for collagen type I. Furthermore, histological and micro-computed tomography analysis showed mineral deposition within BMSC seeded constructs was suppressed by the application of DC. Therefore, while the application of DC in vitro may only lead to modest improvements in the mechanical functionality of cartilaginous grafts, it may play an important role in the development of phenotypically stable constructs.

Background The study aimed to evaluate the combined effect of Pulsed Electromagnetic Field (PEMF) biophysical stimulation and bone marrow concentrate (BMC) in osteochondral defect healing in comparison to the treatment with scaffold alone. Methods An osteochondral lesion of both knees was performed in ten rabbits. One was treated with a collagen scaffold alone and the other with scaffold seeded with BMC. Half of the animals were stimulated by PEMFs (75 Hz, 1.5 mT, 4 h/day) and at 40 d, macroscopic, histological and histomorphometric analyses were performed to evaluate osteochondral defect regeneration. Results Regarding cartilage, the addition of BMC to the scaffold improved cell parameters and the PEMF stimulation improved both cell and matrix parameters compared with scaffold alone. The combination of BMC and PEMFs further improved osteochondral regeneration: there was an improvement in macroscopic, cartilage cellularity and matrix parameters and a reduction in the percentage of cartilage under the tidemark. Epiphyseal bone healing improved in all the osteochondral defects regardless of treatment, although PEMFs alone did not significantly improve the reconstruction of subchondral bone in comparison to treatment with scaffold alone. Conclusions Results show that BMC and PEMFs might have a separate effect on osteochondral regeneration, but it seems that they have a greater effect when used together. Biophysical stimulation is a non-invasive therapy, free from side effects and should be started soon after BMC transplantation to increase the quality of the regenerated tissue. However, because this is the first explorative study on the combination of a biological and a biophysical treatment for osteochondral regeneration, future preclinical and clinical research should be focused on this topic to explore mechanisms of action and the correct clinical translation.

The use of biophysical stimulation, with low-frequency pulsed electromagnetic fields, to modulate osteogenetic response and favour fracture healing has been the subject of wide-ranging research. Current orthopaedics reviews the different modalities of biophysical treatment in search of solutions most adequate to the pathology, the characteristics of the fracture and those of the patient. It is up to the orthopaedist to assess whether the biomechanical conditions of stability of the fracture site are such as not to jeopardize the osteogenetic process. Moreover, experimental studies on articular cartilage have demonstrated that low-frequency pulsed electromagnetic fields control inflammation, protect extracellular matrix and favour chondrocytes metabolic activity. The results of two level I clinical study on patients underwent arthroscopic procedures demonstrated that low-frequency pulsed electromagnetic fields favour patients' recovery both in the short (90 days) and in the long term (3 years). The long term benefit results from biophysical chondroprotection of articular cartilage and from prevention of the fibrotic stimuli exerted by pro-inflammatory cytokines on wound tissue. Biophysical stimulation plays a central role also in regenerative medicine, protecting the repair of tissue from catabolic effects of the inflammatory reaction elicited by the surgical implantation procedure.

Tendons and ligaments (TL) have poor healing capability, and for serious injuries like tears or ruptures, surgical intervention employing autografts or allografts is usually required. Current tissue replacements are nonideal and can lead to future problems such as high retear rates, poor tissue integration, or heterotopic ossification. Alternatively, tissue engineering strategies are being pursued using biodegradable scaffolds. As tendons connect muscle and bone and ligaments attach bones, the interface of TL with other tissues represent complex structures, and this intricacy must be considered in tissue engineered approaches. In this paper, we review recent biofabrication and signaling strategies for biodegradable polymeric scaffolds for TL interfacial tissue engineering. First, we discuss biodegradable polymeric scaffolds based on the fabrication techniques as well as the target tissue application. Next, we consider the effect of signaling factors, including cell culture, growth factors, and biophysical stimulation. Then, we discuss human clinical studies on TL tissue healing using commercial synthetic scaffolds that have occurred over the past decade. Finally, we highlight the challenges and future directions for biodegradable scaffolds in the field of TL and interface tissue engineering.

Administration of exogenous hyaluronic acid (HA) by liquid jet injection is considered as a beneficial therapy for dermatology conditions. This paper reviews variety of the factors which would optimize the clinical output of hyaluronic acid in this treatment modality. A pneumatically accelerated jet penetrates the epidermis and spherically spreads micro-droplets of HA in the dermis without significant damage to the tissue and blood vessels. Kinetic energy of the jet activates two parallel mechanisms of action-mechanical and biological-which act synergistically to initiate and augment the regenerative effect. Jet-induced micro-trauma stimulates collagen synthesis and tissue repair without inflammation. Aside from the biophysical stimulation of dermal fibroblast, the biomolecular properties of exogenous HA provide excellent clinical results for skin atrophy, remodeling of dermal scarring, and reverse formation of fibrotic tissue. The effect is mediated by HA-specific cell receptors and depends on molecular weight and the rheological properties of HA polymer. Skin mechanical properties play a key role in predicting HA dispersion patterns. Tolerability and safety of the treatment approach are determined by the jet's physical impact on the tissue and/or by the safety profile of the injected material. Although pneumatic jet delivery of a hyaluronic acid has a limited use in clinical practice, this treatment approach has a strong potential for extended implementation in esthetic dermatology. The synergistic mechanism has significant advantages of predictable and rapid clinical outcomes with a low discomfort. Additional well-designed investigations are required for establishing a scientific foundation and guidelines for this treatment modality.

Study design This is an in vitro study of bovine disc cells exposed to pulsed electromagnetic fields. Objective The purpose of the present study was to investigate whether pulsed electromagnetic fields (PEMF) effects on the expression of interleukin-6 (IL-6) expression is mediated by two known inflammation regulators, nuclear factor-κB (NF-κβ) and phosphorylated mitogen-activated protein kinase p38 (p38-MAPK) signaling pathways SUMMARY OF BACKGROUND DATA.: Inflammatory cytokines play a dominant role in the pathogenesis of disc degeneration. Increasing evidence showed that PEMF, a noninvasive biophysical stimulation, can have physiologically beneficial effects on inflammation and tissue repair. Our previous research shows that PEMF treatment can reduce IL-6 expression by intervertebral disc cells. However, the underlying mechanisms of PEMF action are yet to be uncovered. Methods Intervertebral disc nuclear pulposus cells were challenged with interleukin-1α (IL-1α) (for mimicking inflammatory microenvironment) and treated with PEMF simultaneously up to 4 hours. Cells were then collected for NF-κβ and phosphorylated p38-MAPK protein detection with Western blot. Additionally, the RelA (p65) subunit of NF-κβ was examined with immunostaining for assessment of NF-κβ activation. Results As expected, Western blot results showed that both NF-κβ and phosphorylated p38 expression were significantly increased by IL-1α treatment. This induction was significantly inhibited to control condition levels by PEMF treatment. Immunostaining demonstrated similar trends, that PEMF treatment reduced the NF-κβ activation induced by IL-1α exposure. Conclusion Our data indicate that the previously-reported inhibitory effect of PEMF treatment on disc inflammation is mediated by NF-κβ and phosphorylated p38-MAPK signaling pathways. These results further establish PEMFs anti-inflammatory activity, and may inform potential future clinical uses for management of inflammation associated with disc degeneration. Level of evidence N/A.

Cartilage lesions are considered to be the first cause of disability in the active population. Treating cartilage injuries is often a challenge for orthopaedic surgeons, owing to the poor regenerative potential of chondrocytes. Pulsed electromagnetic fields (PEMFs) improve cellular proliferation and migration, and ECM induction. PEMFs during or after surgery for chondral lesion have been demonstrated to be useful in inducing better repair and migration of chondrocytes into the bioscaffolds, and to lead to improvement in patient symptoms after surgery. In this review of the literature, we collected a significative number of studies, which support using biophysical stimulation, in particular PEMFs, as first-line treatment during and after surgery to treat chondral lesion.

Dental implants using titanium have greatly advanced through the improvement of designs and surface treatments. Nonetheless, the anatomical limits and physiological changes of the patient are still regarded as obstacles in increasing the success rate of implants further, even with the enhancement of implant products. So there have been many efforts to overcome these limits. The intrinsic potential for bone regeneration can be stimulated through adjuvant treatments with the continuous improvement of implant properties, and this can play an important role in achieving optimum osseointegration toward peripheral bone tissue and securing ultimate long-term implant stability in standard surgical procedures. For this purpose, various chemical, biological, or biophysical measures were developed such as bone grafts, materials, pharmacological agents, growth factors, and bone formation proteins. The biophysical stimulation of bone union includes non-invasive and safe methods. In the beginning, it was developed as a method to enhance the healing of fractures, but later evolved into Pulsed Electromagnetic Field, Low-Intensity Pulsed Ultrasound, and Low-Level Laser Therapy. Their beneficial effects were confirmed in many studies. This study sought to examine bone-implant union and its latest trend as well as the biophysical stimulation method to enhance the union. In particular, this study suggested the enhancement of the function of cells and tissues under a disadvantageous bone metabolism environment through such adjunctive stimulation. This study is expected to serve as a treatment guideline for implant-bone union under unfavorable circumstances caused by systemic diseases hampering bone metabolism or the host environment.

Bioreactors are commonly used to apply biophysically-relevant stimulations to tissue-engineered constructs in order to explore how these stimuli influence tissue development, healing, and homeostasis. These bioreactors offer great flexibility as key features of the stimuli (e.g., duty cycle, frequency, amplitude, duration) can be controlled to elicit a desired cellular response. Controlled delivery of mechanical and/or electrical stimulation has been shown to improve the structure and function of engineered tissue constructs, compared to unstimulated controls, for applications in various musculoskeletal soft tissues. However, most bioreactors that apply mechanical and electrical stimulations, do so to a scaffold after the construct has developed, preventing study of the influence these stimuli have on early construct development. Thus, there is a need for a bioreactor that allows the direct application of mechanical and electrical stimulation to constructsas they develop, to enable such exploration and to better mimic key aspects of tissue development. Hence, the objective of this study was to develop and calibrate a bioreactor to deliver precise mechanical and electrical stimulation, either independently or in combination, to developing scaffold-free tissue constructs. Standard Flexcell Tissue Train plates were modified with stainless steel loading pins and stimulating electrodes to integrate direct mechanical and electrical stimulation, respectively, into our established scaffold-free, single-fiber engineering platform. Linear calibration curves were established, then used to apply precise dynamic mechanical and electrical stimulations, over a range of physiologically relevant strains (0.50, 0.70, 0.75, 1.0, 1.5%) and voltages (1.5, 3.5 V), respectively. Once calibrated, applied mechanical and electrical stimulations were not statistically different from their desired target values, and were consistent whether delivered independently or in combination. Indeed, concurrent delivery of mechanical and electrical stimulation resulted in a negligible change in mechanical (< 2%) and electrical (IMPACT STATEMENTAs the importance of biophysical stimulation in tissue engineering continues to be recognized and incorporated, this bioreactor provides a platform to further our understanding of the roles independent or combined mechanical and electrical stimulation have in tissue development and functional maturation, and may inform future tissue engineering approaches for clinical applications.

Articular cartilage consists of hyaline cartilage, is a major constituent of the human musculoskeletal system and has critical functions in frictionless joint movement and articular homoeostasis. Osteoarthritis (OA) is an inflammatory disease of articular cartilage, which promotes joint degeneration. Although it affects millions of people, there are no satisfying therapies that address this disease at the molecular level. Therefore, tissue regeneration approaches aim at modifying chondrocyte biology to mitigate the consequences of OA. This requires appropriate biochemical and biophysical stimulation of cells. Regarding the latter, mechanotransduction of chondrocytes and their precursor cells has become increasingly important over the last few decades. Mechanotransduction is the transformation of external biophysical stimuli into intracellular biochemical signals, involving sensor molecules at the cell surface and intracellular signalling molecules, so-called mechano-sensors and -transducers. These signalling events determine cell behaviour. Mechanotransducing ion channels and gap junctions additionally govern chondrocyte physiology. It is of great scientific and medical interest to induce a specific cell behaviour by controlling these mechanotransduction pathways and to translate this knowledge into regenerative clinical therapies. This review therefore focuses on the mechanotransduction properties of integrins, cadherins and ion channels in cartilaginous tissues to provide perspectives for cartilage regeneration.

In cell-based therapies for cartilage lesions, the main problem is still the formation of fibrous cartilage, caused by underlying de-differentiation processes ex vivo. Biophysical stimulation is a promising approach to optimize cell-based procedures and to adapt them more closely to physiological conditions. The occurrence of mechano-electrical transduction phenomena within cartilage tissue is physiological and based on streaming and diffusion potentials. The application of exogenous electric fields can be used to mimic endogenous fields and, thus, support the differentiation of chondrocytes in vitro. For this purpose, we have developed a new device for electrical stimulation of chondrocytes, which operates on the basis of capacitive coupling of alternating electric fields. The reusable and sterilizable stimulation device allows the simultaneous use of 12 cavities with independently applicable fields using only one main supply. The first parameter settings for the stimulation of human non-degenerative chondrocytes, seeded on collagen type I elastin-based scaffolds, were derived from numerical electric field simulations. Our first results suggest that applied alternating electric fields induce chondrogenic re-differentiation at the gene and especially at the protein level of human de-differentiated chondrocytes in a frequency-dependent manner. In future studies, further parameter optimizations will be performed to improve the differentiation capacity of human cartilage cells.

Objectives To primarily assess the existing literature about Magnetic Resonance Therapy (MRT) or Molecular Biophysical Stimulation Therapy (MBST) in the treatment of patients with osteoarthritis (OA). The scoping review question was: What has been reported about MRT or MBST concerning treatment of patients with OA? Key findings The applied treatment program consisted of one hour daily treatment for patients in all the included studies. In terms of duration of treatment, four studies suggested treatment for nine consecutive days, two for five days and one study reported treatment on weekdays for two weeks. Six of the studies investigated the effect of MRT on the knee and one study for finger, ankle, and hip, respectively. Consensus across studies was that MRT had a positive, almost always significant, effect. Six out of the seven studies had subjective outcome measurements such as pain, quality of life and joint function, which were measured through self-reported questionnaires. One study combined ultrasonography with Magnetic Resonance Imaging (MRI) to evaluate structural joint changes. This evaluation was performed by a radiologist. One study used objective measurement of cartilage thickness through a minimal distance algorithm. All tests used MBST-systems. Conclusion This scoping review showed that there seems to be a beneficial effect of MRT in the treatment of patients with OA in relation to improvement in pain, joint function, and quality of life. However, more robust research and further evaluation of MRT are needed. Implications for practice Treating patients diagnosed with OA with MRT for one hour for five to ten days seemed to improve pain, joint function, quality of life as well as regeneration of cartilage. However, limitations of the included studies in this scoping review, such as a general lack of control groups, low sample sizes, lack of control for confounding factors such as medication, calls for more robust research with stronger study designs.

Due to the ability to differentiate into variety of cell types, mesenchymal stem cells (MSCs) hold promise as source in cell-based therapy for treating injured tissue and degenerative diseases. The potential use of MSCs to replace or repair damaged tissues may depend on the efficient differentiation protocols to derive specialized cells without any negative side effects. Identification of appropriate cues that support the lineage-specific differentiation of stem cells is critical for tissue healing and cellular therapy. Recently, a number of stimuli have been utilized to direct the differentiation of stem cells. Biochemical stimuli such as small molecule, growth factor and miRNA have been traditionally used to regulate the fate of stem cells. In recent years, many studies have reported that biophysical stimuli including cyclic mechanical strain, fluid shear stress, microgravity, electrical stimulation, matrix stiffness and topography can also be sensed by stem cells through mechanical receptors, thus affecting the stem cell behaviors including their differentiation potential. In this paper, we review all the most recent literature on the application of biochemical and biophysical cues on regulating MSC differentiation. An extensive literature search was done using electronic database (Medline/Pubmed). Although there are still some challenges that need to be taken into consideration before translating these methods into clinics, biochemical and biophysical stimulation appears to be an attractive method to manipulate the lineage commitment of MSCs.

Most kidney diseases begin with abnormalities in glomerular podocytes, motivating the need for podocyte models to study pathophysiological mechanisms and new treatment options. However, podocytes cultured in vitro face a limited ability to maintain appreciable extents of differentiation hallmarks, raising concerns over the relevance of study results. Many key properties such as nephrin expression and morphology reach plateaus that are far from the in vivo levels. Here, we demonstrate that a biomimetic topography, consisting of microhemispheres arrayed over the cell culture substrate, promotes podocyte differentiation in vitro. We define new methods for fabricating microscale curvature on various substrates, including a thin porous membrane. By growing podocytes on our topographic substrates, we found that these biophysical cues augmented nephrin gene expression, supported full-size nephrin protein expression, encouraged structural arrangement of F-actin and nephrin within the cell, and promoted process formation and even interdigitation compared to the flat substrates. Furthermore, the topography facilitated nephrin localization on curved structures while nuclei lay in the valleys between them. The improved differentiation was also evidenced by tracking barrier function to albumin over time using our custom topomembranes. Overall, our work presents accessible methods for incorporating microcurvature on various common substrates, and demonstrates the importance of biophysical stimulation in supporting higher-fidelity podocyte cultivation in vitro.

Modern physical therapy could be conducted in many ways. Some of them are based on methods using the biophysical stimulation with a time-varying magnetic field. Although there are many devices functioning on this principle, the effect of the magnetic field remains arguable. Nowadays, the trend is to use low-frequency electromagnetic fields which allow an induced electric current to form in the exposed tissue. However, the use of such magnetic fields is associated with certain risks. It is questionable whether the application of such magnetic fields in certain locations might affect physiological electrical activity concerning the fact that the electrical current is generated in the tissue. For this reason, this paper aimed to monitor the effect of high-induction magnetic stimulation in an area where the electrical conduction system of the heart could be affected. Although no pathological signs in the ECG record were observed by the supervising physician, the results show that after the application of high-induction magnetic stimulation, the variability of the heart rhythm is affected since statistically significant differences were observed in the values of parameters describing the variability, i.e. SDNN (p = 0.040) and std HR (p = 0.004), pre and post the HIMS application.

Engineered skeletal muscle grafts may be employed in various applications including the treatment of volumetric muscle loss (VML) and pharmacological drug screening. To recapitulate the well-defined structure of native muscle, tensile strains have been applied to the grafts. In this study, we cultured C2C12 murine myoblasts on electrospun fibrin microfiber bundles for 7 days in custom-built bioreactor units and investigated the impact of strain regimen and delayed onset of tensile straining on myogenic outcomes. The substrate topography induced uniaxial alignment of cells in all (strained and unstrained) groups. The engineered grafts in strained groups were subjected to 10% strain amplitude for 6 h per day. We found that both static and cyclic uniaxial strains resulted in similar morphological and gene expression outcomes. However, relative to 0% strain groups, there were stark increases in myotube diameter, myosin heavy chain (MHC) coverage, and expression of key myogenic genes (Pax 7, Troponin, MHC I, MHC IIb, MHC IIx) only if strain was applied at Days 5–7 rather than Days 3–7. This finding suggests that a critical indicator of myogenic improvement under strain in our system is the phenotype of the cells at the onset of strain and suggests that this is a key parameter that should be considered in studies where myoblasts are subjected to biophysical stimulation to promote tissue formation. Statement of Significance This is the first report on the impact of the timing of the initial application of mechanical strain for improving the myogenic outcomes of 3D engineered skeletal muscle grafts. In this work, immature skeletal myoblasts were grown on topographically aligned, electrospun fibrin microfiber bundles and we applied 10% uniaxial static or cyclic strain. We concluded that the maturity of myoblasts prior to strain application, rather than strain waveform, was the primary predictor of improved myogenic outcomes, including myogenic gene expression and myotube morphology. Elucidating the optimal conditions for strain application is a vital step in recapitulating physiological myogenic properties in tissue engineered skeletal muscle constructs, with applications for treating volumetric muscle loss, disease modeling, and drug testing.

Periodontal ligament stem cells (PDLSCs) have been confirmed to have self-renewal capacity and multidifferentiation potential and are good candidates for periodontal tissue regeneration. Pulsed electromagnetic field (PEMF) has been demonstrated to promote osteogenesis in non-union fractures, partly by regulating mesenchymal stem cells or osteoblast activity. However, there is no report about the osteo-inductive effect of PEMF stimulation on human PDLSCs (hPDLSCs). Thus, we tested the hypothesis that PEMF biophysical stimulation alone has an influence on the proliferation and osteogenic differentiation of hPDLSCs. To detect the osteo-inductive potential of bone morphogenetic protein (BMP9), we transfected the STRO-1(+) /CD146(+) hPDLCSs with BMP9-expressing recombinant adenoviruses. We examined the proliferation and osteogenic differentiation of hPDLSCs treated with either PEMF (15 Hz, 1 h daily, different intensities), or BMP9, or both stimuli. Cell counting kit-8 (CCK-8) assay showed that PEMF of different intensities had no effect on the proliferation of hPDLSCs and did not enhance the proliferative capability of BMP9-transfected cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting showed that the combination of both PEMFs (1.8 or 2.4 mT) and BMP9 stimulation had a synergistic effect on early and intermediate osteogenic genes and protein expressions of runt-related transcription factor 2, alkaline phosphatase, osteopontin, and late mineralized extracellular matrix formation in hPDLSCs. Bioelectromagnetics. 38:63-77, 2017. © 2016 Wiley Periodicals, Inc.

Bone defects caused by trauma and surgery are common clinical problems encountered by orthopedic surgeons. Thus, a hard-textured, natural-like biomaterial that enables encapsulated cells to obtain the much-needed biophysical stimulation and produce functional bone tissue is needed. Incorporating nanomaterials into cell-laden hydrogels is a straightforward tactic for producing tissue engineering structures that integrate perfectly with the body and for tailoring the material characteristics of hydrogels without hindering nutrient exchange with the surroundings. In this review, recent developments in inorganic nanocomposite hydrogels for bone tissue engineering that are of vital importance but have not yet been comprehensively reviewed are summarized.

Diaphyseal fractures represent a complex biological entity that could often end into impaired bone-healing, with delayed union and non-union occurring up to 10% of cases. The role of the modern orthopaedic surgeon is to optimize the fracture healing environment, recognize and eliminate possible interfering factors, and choose the best suited surgical fixation technique. The impaired reparative process after surgical intervention can be modulated with different surgical techniques, such as dynamization or exchange nailing after failed intramedullary nailing. Moreover, the mechanical stability of a nail can be improved through augmentation plating, bone grafting or external fixation techniques with satisfactory results. According to the “diamond concept”, local therapies, such as osteoconductive scaffolds, bone growth factors, and osteogenic cells can be successfully applied in “polytherapy” for the enhancement of delayed union and non-union of long bones diaphyseal fractures. Moreover, systemic anti-osteoporosis anabolic drugs, such as teriparatide, have been proposed as off-label treatment for bone healing enhancement both in fresh complex shaft fractures and impaired unions, especially for fragility fractures. The article aims to review the biological and mechanical principles of failed reparative osteogenesis of diaphyseal fractures after surgical treatment. Moreover, the evidence about the modern non-surgical and pharmacological options for bone healing enhancement will discussed., Highlights • Bone healing of shaft fractures depends on mechanical and biological factors • Surgical treatments aim to restore the mechanical stability at fracture site • Biological treatments comprehend scaffolds, growth factors and cell therapies • No evidence supports biophysical stimulation (PEMF, LIPUS) for acute shaft fractures • Poor evidence exists supporting anabolic agents therapies for acute shaft fractures • The use of multiple combined treatments (polytherapy) provides higher union rates

Tendon-healing capacity is limited due to its hypocellularity and hypovascularity even after surgical repair. Tissue engineering plays a promising role in optimizing tendon regeneration. In this chapter, we discuss tendon macroscopic and microscopic structures, the native healing cascade, and the current advances and challenges of tendon tissue engineering. These engineering principles are split into three fundamental factors: cells, scaffolds, and growth factors. Several cell sources are described for clinical application, including various kinds of stem cells. We then explore the benefits and advantages of biological scaffolds over synthetic scaffolds. Next, we discuss several growth factors that play important roles in the tendon-healing process that have augmented tendon healing in animal models. Moreover, biophysical stimulation and drug repositioning concepts are described as possible strategies to facilitate tendon regeneration.

Mesenchymal stem cells (MSCs) provide a promising source for cartilage tissue regeneration strategies. The use of MSCs for such strategies, however, remains challenging due to the low targeting and low chondrogenic differentiation efficiency of these cells to the desired site. In an attempt to overcome such problems, we propose the use of a magnetoresponsive stem cell spheroid (MR-SCS)-based cartilage recovery platform that allows for precise targeting using an electromagnetic actuation (EMA) system to provide magnetic control and low-frequency electromagnetic field (LF-EMF) to allow for biophysical stimulation to promote chondrogenic differentiation. MR-SCSs were fabricated from mouse-derived MSCs that were labeled with magnetic nanoparticles (MNPs) using 3D culture methods, and these particles exhibited no cytotoxicity and did not affect chondrogenic differentiation. Locomotion of MR-SCS that was mediated by the EMA system was successfully demonstrated in 3D phantom and ex vivo models. Additionally, LF-EMF stimulation of MR-SCS resulted in increased expression levels of cartilage specific markers, collagen type II, SOX9, and Aggrecan. Finally, histological evaluation revealed an apparent improvement in the regeneration of cartilage tissue in an ex vivo model of the porcine femur in response to LF-EMF stimulation. These results suggest that our experimental platform consisting of MR-SCSs that are subjected to EMA and LF-EMF stimulation may provide a promising therapeutic system for cartilage tissue regeneration.