Mesenchymal stem cells (MSCs) can differentiate into multiple different tissue lineages and have favourable immunogenic potential making them an attractive prospect for regenerative medicine. As an ...essential part of the manufacturing process, preservation of these cells whilst maintaining potential is of critical importance. An uncontrolled area of storage remains the rate of change of temperature during freezing and thawing. Controlled-rate freezers attempted to rectify this; however, the change of phase from liquid to solid introduces two extreme phenomena; a rapid rise and a rapid fall in temperature in addition to the intended cooling rate (normally -1 °C/min) as a part of the supercooling event in cryopreservation. Nucleation events are well known to initiate the freezing transition although their active use in the form of ice nucleation devices (IND) are in their infancy in cryopreservation. This study sought to better understand the effects of ice nucleation and its active instigation with the use of an IND in both a standard cryotube with MSCs in suspension and a high-throughput adhered MSC 96-well plate set-up. A potential threshold nucleation temperature for best recovery of dental pulp MSCs may occur around -10 °C and for larger volume cell storage, IND and fast thaw creates the most stable process. For adhered cells, an IND with a slow thaw enables greatest metabolic activity post-thaw. This demonstrates a necessity for a medical grade IND to be used in future regenerative medicine manufacturing with the parameters discussed in this study to create stable products for clinical cellular therapies.
There is a current need for a therapy that can alleviate the social and economic burden that presents itself with debilitating and recurring musculoskeletal soft tissue injuries and disorders. ...Currently, several therapies are emerging and undergoing trials in animal models; these focus on the manipulation and administration of several growth factors implicated with healing. However, limitations include in vivo instability, reliance on biocompatible and robust carriers and restricted application procedures (local and direct). The aim of this paper is therefore to critically review the current literature surrounding the use of BPC 157, as a feasible therapy for healing and functional restoration of soft tissue damage, with a focus on tendon, ligament and skeletal muscle healing. Currently, all studies investigating BPC 157 have demonstrated consistently positive and prompt healing effects for various injury types, both traumatic and systemic and for a plethora of soft tissues. However, to date, the majority of studies have been performed on small rodent models and the efficacy of BPC 157 is yet to be confirmed in humans. Further, over the past two decades, only a handful of research groups have performed in-depth studies regarding this peptide. Despite this, it is apparent that BPC 157 has huge potential and following further development has promise as a therapy to conservatively treat or aid recovery in hypovascular and hypocellular soft tissues such as tendon and ligaments. Moreover, skeletal muscle injury models have suggested a beneficial effect not only for disturbances that occur as a result of direct trauma but also for systemic insults including hyperkalamia and hypermagnesia. Promisingly, there are few studies reporting any adverse reactions to the administration of BPC 157, although there is still a need to understand the precise healing mechanisms for this therapy to achieve clinical realisation.
Purpose of Review
The treatment of non-union fractures represents a significant challenge for orthopaedic surgeons. In recent years, biologic agents have been investigated and utilised to support and ...improve bone healing. Among these agents, platelet-rich plasma (PRP) is an emerging strategy that is gaining popularity. The aim of this review is to evaluate the current literature regarding the application and clinical effectiveness of PRP injections, specifically for the treatment of non-union fractures.
Recent Findings
The majority of published studies reported that PRP accelerated fracture healing; however, this evidence was predominantly level IV. The lack of randomised, clinical trials (level I–II evidence) is currently hampering the successful clinical translation of PRP as a therapy for non-union fractures. This is despite the positive reports regarding its potential to heal non-union fractures, when used in isolation or in combination with other forms of treatment.
Summary
Future recommendations to facilitate clinical translation and acceptance of PRP as a therapy include the need to investigate the effects of administering higher volumes of PRP (i.e. 5–20 mL) along with the requirement for more prolonged (> 11 months) randomised clinical trials.
The widening gap between organ availability and need is resulting in a worldwide crisis, particularly concerning kidney transplantation. Regenerative medicine options are becoming increasingly ...advanced and are taking advantage of progress in novel manufacturing techniques, including 3D bioprinting, to deliver potentially viable alternatives. Cell-integrated and wearable artificial kidneys aim to create convenient and efficient systems of filtration and restore elements of immunoregulatory function. Whilst preliminary clinical trials demonstrated promise, manufacturing and trial design issues and identification of suitable and sustainable cell sources have shown that more development is required for market progression. Tissue engineering and advances in biomanufacturing techniques offer potential solutions for organ shortages; however, due to the complex kidney structure, previous attempts have fallen short. With the recent development and progression of 3D bioprinting, cell positioning and resolution of material deposition in organ manufacture have never seen greater control. Cell sources for constructing kidney building blocks and populating both biologic and artificial scaffolds and matrices have been identified, but in vitro culturing and/or differentiation, in addition to maintaining phenotype and viability during and after lengthy and immature manufacturing processes, presents additional problems. For all techniques, significant process barriers, clinical pathway identification for translation of models to humans, scaffold material availability, and long-term biocompatibility need to be addressed prior to clinical realisation.
Three‐dimensional tissue‐engineered structures enable more representative determination of novel drug or material effects on tissue than traditional monolayer cell cultures. This study sought to ...better understand how key manufacturing variables affect the myotube characteristics of a skeletal muscle model toward reducing resource use and to develop an understanding of scaling on model consistency. C2C12 murine myoblasts were seeded in a tethered collagen scaffold from which directional myotubes form in response to lines of tension and a change in medium. Collagen polymerizing area length‐to‐width ratios greater than one were found to reduced cell–matrix attachment and remodeling forces significantly (p < .05) correlating to a reduction in cell fusion potential. Following this, utilizing a factorial design of experiment, 4 million C2C12s/ml, with a polymerizing area width 150% of the anchor point, produced the most favorable myotube characteristics and dramatically reduced the incidence of rupture. Scaled constructs showed no significant differences when compared to larger models. Approximately 20 myotubes with a variation in the alignment of <25° in the central region were consistently observed in the final models. This demonstrates the influence of initial manufacturing variables on tissue formation and has produced a benchmark model for consistent production across scaled constructs for future optimization and as a potential cost‐effective preclinical testbed.
Three‐dimensional tissue‐engineered structures enable more representative determination of novel drug or material effects on tissue than traditional monolayer cell cultures. This study sought to better understand how key manufacturing variables affect the myotube characteristics of a skeletal muscle model toward reducing resource use and to develop an understanding of scaling on model consistency.
In vitro 3D tissue‐engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than ...animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co‐culture of TE skeletal muscle and bone are investigated. High‐glucose Dulbecco's modified Eagle medium (HG‐DMEM) supplemented with 20% fetal bovine serum followed by HG‐DMEM with 2% horse serum is found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an upregulation of RUNX2/CBFa1 in TE85s. Myotube formation is also evident within indirect contact monolayer cultures. Finally, in 3D co‐cultures, TE85 collagen/hydroxyapatite constructs have significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen‐based C2C12 skeletal muscle constructs; however, fusion within these constructs appears reduced. This work demonstrates the first report of the simultaneous co‐culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step toward a full in vitro 3D musculoskeletal junction model.
Laboratory‐based 3D models of individual tissues have been shown to better represent native tissue than single layer cell culture. In this study, the conditions to form both skeletal muscle and bone constructs are demonstrated. Indicators of osteogenic differentiation are found to increase in the presence of skeletal muscle. In contrast, skeletal muscle myogenesis is reduced.
The prevalence of the rotator cuff (RC) tears is ~ 21% in the general population, with higher incidences in individuals over 50. Irrespective of surgical repair techniques employed, re-tear rates are ...alarmingly high, indicating the need for improvement to the current treatment methods. A method that has recently increased in popularity is the administration of platelet-rich-plasma (PRP), as it has been proposed to significantly encourage and improve healing in a plethora of musculoskeletal tissues, although experimental conditions and results are often variable. This review aims to critically evaluate current literature concerning the use of PRP, specifically for the treatment of RC tears. There are ongoing conflicts debating the effectiveness of PRP to treat RC tears; with literature both in favour and against its use either having profound methodological weaknesses and/or limited applicability to most individuals with RC tears. There are numerous factors that may influence effectiveness, including the subgroup of patients studied and the timing and method of PRP delivery. Thus, in order to ascertain the clinical effectiveness of PRP for RC tears, the preparation protocol and composition of PRP must be standardised, so an accurate assessment and comparisons can be undertaken. Prior to clinical realisation, there is a requirement for a defined, standardised, quality-controlled protocol/procedure considering composition/formulation (of PRP); injury severity, dosage, frequency, timings, controls used, patient group, and rehabilitation programmes. Nevertheless, it is concluded that the initial step to aid the progression of PRP to treat RC tears is to standardise its preparation and delivery.
Rotator cuff injuries (RCIs) present a major health problem due to high incidences of degenerative tears greater than 3 cm and prevalence of re-tears following surgical procedures. Since healing and ...functional restoration relies upon bone ingrowth into the tendon, it is hypothesised that sustained delivery of osteoinductive factors including bone morphogenetic proteins (BMPs), specifically BMP2–7, may significantly improve RCI tendon-bone healing. Here, growth factor candidates and delivery mechanisms are reviewed, specifically for improved RCI healing through enhanced bone ingrowth. In addition to BMPs, other potentially osteogenic factors including platelet-derived growth factors (PDGF), fibroblast growth factor (FGF), transforming growth beta isoforms (TGF-β1 and TGF-3) and parathyroid hormone (PTH) are evaluated since they can induce bone formation at the healing tendon attachment site. Several challenges must be addressed prior to clinical translation. The majority of published studies utilise in vivo animal models. In general, BMP-7 demonstrates a stronger stimulating effect when compared to BMP-2; the reported effectiveness of BMP-2 is often conflicting. Alternative factors, including PDGF and PTH, also demonstrate potential for assisting bone growth in enthesis healing. The use of sustained and biomimetic delivery systems appears to have the greatest positive effects. Some studies have demonstrated a dose-dependent effect, in conjunction with varying age, indicating that stratified therapies could be a viable solution for RCI healing. To adequately resolve potential treatments for RCI, further expanded and correlated animal trials must be undertaken, and indicative human trials are required with consideration of surgical and patient-specific influences.
Pre-clinical studies are a necessary step in the process of material and drug testing. For this, high-throughput monolayer cell cultures are conducted followed by in vivo animal experiments. However, ...animal use is ethically questionable and in many cases yields misleading results. In vitro three dimensional (3D) tissue engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer cultures and are less ethically questionable than animal models. Therefore, an in vitro biomimetic musculoskeletal junction (MSKjct) is required as a more relevant pre-clinical testbed. This thesis describes the steps taken to co-culture 3D TE skeletal muscle and bone models as a material testbed and towards an in vitro MSKjct.
Histone lysine methylation, mediated by mixed-lineage leukemia (MLL) proteins, is now known to be critical in the regulation of gene expression, genomic stability, cell cycle and nuclear ...architecture. Despite MLL proteins being postulated as essential for normal development, little is known about the specific functions of the different MLL lysine methyltransferases. Here we report heterozygous variants in the gene KMT2B (also known as MLL4) in 27 unrelated individuals with a complex progressive childhood-onset dystonia, often associated with a typical facial appearance and characteristic brain magnetic resonance imaging findings. Over time, the majority of affected individuals developed prominent cervical, cranial and laryngeal dystonia. Marked clinical benefit, including the restoration of independent ambulation in some cases, was observed following deep brain stimulation (DBS). These findings highlight a clinically recognizable and potentially treatable form of genetic dystonia, demonstrating the crucial role of KMT2B in the physiological control of voluntary movement.
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