Objectives. The role of mechanical stress and
Aims. Osteoarthritis (OA) is a common degenerative joint disease. The osteocyte transcriptome is highly relevant to osteocyte biology. This study aimed to explore the osteocyte transcriptome in subchondral bone affected by OA. Methods. Gene expression profiles of OA subchondral bone were used to identify disease-relevant genes and signalling pathways. RNA-sequencing data of a bone loading model were used to identify the loading-responsive gene set. Weighted gene co-expression network analysis (WGCNA) was employed to develop the osteocyte mechanics-responsive gene signature. Results. A group of 77 persistent genes that are highly relevant to extracellular matrix (ECM) biology and bone remodelling signalling were identified in OA subchondral lesions. A loading responsive gene set, including 446 principal genes, was highly enriched in OA medial tibial plateaus compared to lateral tibial plateaus. Of this gene set, a total of 223 genes were identified as the main contributors that were strongly associated with osteocyte functions and signalling pathways, such as ECM modelling, axon guidance, Hippo, Wnt, and
Aims. Proliferation, migration, and differentiation of anterior cruciate ligament (ACL) remnant and surrounding cells are fundamental processes for ACL reconstruction; however, the interaction between ACL remnant and surrounding cells is unclear. We hypothesized that ACL remnant cells preserve the capability to regulate the surrounding cells’ activity, collagen gene expression, and tenogenic differentiation. Moreover, extracorporeal shock wave (ESW) would not only promote activity of ACL remnant cells, but also enhance their paracrine regulation of surrounding cells. Methods. Cell viability, proliferation, migration, and expression levels of Collagen-I (COL-I) A1,
Aims. Developmental dysplasia of the hip (DDH) is a complex musculoskeletal disease that occurs mostly in children. This study aimed to investigate the molecular changes in the hip joint capsule of patients with DDH. Methods. High-throughput sequencing was used to identify genes that were differentially expressed in hip joint capsules between healthy controls and DDH patients. Biological assays including cell cycle, viability, apoptosis, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were performed to determine the roles of the differentially expressed genes in DDH pathology. Results. More than 1,000 genes were differentially expressed in hip joint capsules between healthy controls and DDH. Both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that extracellular matrix (ECM) modifications, muscle system processes, and cell proliferation were markedly influenced by the differentially expressed genes. Expression of Collagen Type I Alpha 1 Chain (COL1A1), COL3A1, matrix metalloproteinase-1 (MMP1), MMP3, MMP9, and MMP13 was downregulated in DDH, with the loss of collagen fibres in the joint capsule. Expression of
Objectives. The injured anterior cruciate ligament (ACL) is thought to exhibit an impaired healing response, and attempts at surgical repair have not been successful. Connective tissue growth factor (CTGF) is reported to be associated with wound healing, probably through
The formation of restrictive adhesions around the musculotendinous unit after injury is one of the most vexing processes faced by the surgeon. In flexor tendons it has been shown that the synovial tissue is the source of aggressive fibroblasts which contribute to this process. Using a rabbit model, we have examined the effects of treating the synovial sheath with the antimetabolite 5-fluorouracil (5-FU) for five minutes. Inflammatory, proliferative and molecular markers were compared in the response of the treated and control tendons to injury. Compared with a control group we found that the proliferative and inflammatory responses were significantly reduced (p <
0.001) in the treated tendons. Not only was there a reduction in the cellular and cytokine response, but there also was a significant (p <
0.001) reduction in the level of activity of the known pro-scarring agent,
Dupuytren’s contracture is characterized by increased fibrosis of the palmar aponeurosis, with eventual replacement of the surrounding fatty tissue with palmar fascial fibromatosis. We hypothesized that adipocytokines produced by adipose tissue in contact with the palmar aponeurosis might promote fibrosis of the palmar aponeurosis. We compared the expression of the adipocytokines adiponectin and leptin in the adipose tissue surrounding the palmar aponeurosis of male patients with Dupuytren’s contracture, and of male patients with carpal tunnel syndrome (CTS) as the control group. We also examined the effects of adiponectin on fibrosis-related genes and proteins expressed by fibroblasts in the palmar aponeurosis of patients with Dupuytren’s contracture.Aims
Methods
Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes – the main cellular components in BMAC – interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes. Cite this article:
Exosomes (exo) are involved in the progression of osteoarthritis (OA). This study aimed to investigate the function of dysfunctional chondrocyte-derived exo (DC-exo) on OA in rats and rat macrophages. Rat-derived chondrocytes were isolated, and DCs induced with interleukin (IL)-1β were used for exo isolation. Rats with OA (n = 36) or macrophages were treated with DC-exo or phosphate-buffered saline (PBS). Macrophage polarization and autophagy, and degradation and chondrocyte activity of cartilage tissues, were examined. RNA sequencing was used to detect genes differentially expressed in DC-exo, followed by RNA pull-down and ribonucleoprotein immunoprecipitation (RIP). Long non-coding RNA osteoarthritis non-coding transcript (OANCT) and phosphoinositide-3-kinase regulatory subunit 5 (PIK3R5) were depleted in DC-exo-treated macrophages and OA rats, in order to observe macrophage polarization and cartilage degradation. The PI3K/AKT/mammalian target of rapamycin (mTOR) pathway activity in cells and tissues was measured using western blot.Aims
Methods
Cite this article:
This study aimed to demonstrate the promoting effect of elastic fixation on fracture, and further explore its mechanism at the gene and protein expression levels. A closed tibial fracture model was established using 12 male Japanese white rabbits, and divided into elastic and stiff fixation groups based on different fixation methods. Two weeks after the operation, a radiograph and pathological examination of callus tissue were used to evaluate fracture healing. Then, the differentially expressed proteins (DEPs) were examined in the callus using proteomics. Finally, in vitro cell experiments were conducted to investigate hub proteins involved in this process.Aims
Methods
To determine whether platelet-rich plasma (PRP) injection improves outcomes two years after acute Achilles tendon rupture. A randomized multicentre two-arm parallel-group, participant- and assessor-blinded superiority trial was undertaken. Recruitment commenced on 28 July 2015 and two-year follow-up was completed in 21 October 2019. Participants were 230 adults aged 18 years and over, with acute Achilles tendon rupture managed with non-surgical treatment from 19 UK hospitals. Exclusions were insertion or musculotendinous junction injuries, major leg injury or deformity, diabetes, platelet or haematological disorder, medication with systemic corticosteroids, anticoagulation therapy treatment, and other contraindicating conditions. Participants were randomized via a central online system 1:1 to PRP or placebo injection. The main outcome measure was Achilles Tendon Rupture Score (ATRS) at two years via postal questionnaire. Other outcomes were pain, recovery goal attainment, and quality of life. Analysis was by intention-to-treat.Aims
Methods
The lack of disease-modifying treatments for osteoarthritis (OA) is linked to a shortage of suitable biomarkers. This study combines multi-molecule synovial fluid analysis with machine learning to produce an accurate diagnostic biomarker model for end-stage knee OA (esOA). Synovial fluid (SF) from patients with esOA, non-OA knee injury, and inflammatory knee arthritis were analyzed for 35 potential markers using immunoassays. Partial least square discriminant analysis (PLS-DA) was used to derive a biomarker model for cohort classification. The ability of the biomarker model to diagnose esOA was validated by identical wide-spectrum SF analysis of a test cohort of ten patients with esOA.Aims
Methods
Osteoarthritis (OA), one of the most common motor system disorders, is a degenerative disease involving progressive joint destruction caused by a variety of factors. At present, OA has become the fourth most common cause of disability in the world. However, the pathogenesis of OA is complex and has not yet been clarified. Long non-coding RNA (lncRNA) refers to a group of RNAs more than 200 nucleotides in length with limited protein-coding potential, which have a wide range of biological functions including regulating transcriptional patterns and protein activity, as well as binding to form endogenous small interference RNAs (siRNAs) and natural microRNA (miRNA) molecular sponges. In recent years, a large number of lncRNAs have been found to be differentially expressed in a variety of pathological processes of OA, including extracellular matrix (ECM) degradation, synovial inflammation, chondrocyte apoptosis, and angiogenesis. Obviously, lncRNAs play important roles in regulating gene expression, maintaining the phenotype of cartilage and synovial cells, and the stability of the intra-articular environment. This article reviews the results of the latest research into the role of lncRNAs in a variety of pathological processes of OA, in order to provide a new direction for the study of OA pathogenesis and a new target for prevention and treatment. Cite this article:
Transforming growth factor-beta2 (TGF-β2) is recognized as a versatile cytokine that plays a vital role in regulation of joint development, homeostasis, and diseases, but its role as a biological mechanism is understood far less than that of its counterpart, TGF-β1. Cartilage as a load-resisting structure in vertebrates however displays a fragile performance when any tissue disturbance occurs, due to its lack of blood vessels, nerves, and lymphatics. Recent reports have indicated that TGF-β2 is involved in the physiological processes of chondrocytes such as proliferation, differentiation, migration, and apoptosis, and the pathological progress of cartilage such as osteoarthritis (OA) and rheumatoid arthritis (RA). TGF-β2 also shows its potent capacity in the repair of cartilage defects by recruiting autologous mesenchymal stem cells and promoting secretion of other growth factor clusters. In addition, some pioneering studies have already considered it as a potential target in the treatment of OA and RA. This article aims to summarize the current progress of TGF-β2 in cartilage development and diseases, which might provide new cues for remodelling of cartilage defect and intervention of cartilage diseases.
Re-rupture is common after primary flexor tendon repair. Characterization of the biological changes in the ruptured tendon stumps would be helpful, not only to understand the biological responses to the failed tendon repair, but also to investigate if the tendon stumps could be used as a recycling biomaterial for tendon regeneration in the secondary grafting surgery. A canine flexor tendon repair and failure model was used. Following six weeks of repair failure, the tendon stumps were analyzed and characterized as isolated tendon-derived stem cells (TDSCs).Objectives
Methods
The long head of the biceps (LHB) is often resected in shoulder surgery and could therefore serve as a cell source for tissue engineering approaches in the shoulder. However, whether it represents a suitable cell source for regenerative approaches, both in the inflamed and non-inflamed states, remains unclear. In the present study, inflamed and native human LHBs were comparatively characterized for features of regeneration. In total, 22 resected LHB tendons were classified into inflamed samples (n = 11) and non-inflamed samples (n = 11). Proliferation potential and specific marker gene expression of primary LHB-derived cell cultures were analyzed. Multipotentiality, including osteogenic, adipogenic, chondrogenic, and tenogenic differentiation potential of both groups were compared under respective lineage-specific culture conditions.Objectives
Methods
For cementless implants, stability is initially attained by an interference fit into the bone and osteo-integration may be encouraged by coating the implant with bioactive substances. Blood based autologous glue provides an easy, cost-effective way of obtaining high concentrations of growth factors for tissue healing and regeneration with the intention of spraying it onto the implant surface during surgery. The aim of this study was to incorporate nucleated cells from autologous bone marrow (BM) aspirate into gels made from the patient’s own blood, and to investigate the effects of incorporating three different concentrations of platelet rich plasma (PRP) on the proliferation and viability of the cells in the gel. The autologous blood glue (ABG) that constituted 1.25, 2.5, and 5 times concentration PRP were made with and without equal volumes of BM nucleated cells. Proliferation, morphology, and viability of the cells in the glue was measured at days 7 and 14 and compared to cells seeded in fibrin glue.Aims
Methods
Chondrocyte hypertrophy represents a crucial turning point during endochondral bone development. This process is tightly regulated by various factors, constituting a regulatory network that maintains normal bone development. Histone deacetylase 4 (HDAC4) is the most well-characterized member of the HDAC class IIa family and participates in different signalling networks during development in various tissues by promoting chromatin condensation and transcriptional repression. Studies have reported that HDAC4-null mice display premature ossification of developing bones due to ectopic and early-onset chondrocyte hypertrophy. Overexpression of HDAC4 in proliferating chondrocytes inhibits hypertrophy and ossification of developing bones, which suggests that HDAC4, as a negative regulator, is involved in the network regulating chondrocyte hypertrophy. Overall, HDAC4 plays a key role during bone development and disease. Thus, understanding the role of HDAC4 during chondrocyte hypertrophy and endochondral bone formation and its features regarding the structure, function, and regulation of this process will not only provide new insight into the mechanisms by which HDAC4 is involved in chondrocyte hypertrophy and endochondral bone development, but will also create a platform for developing a therapeutic strategy for related diseases.
Experimental studies indicate that non-steroidal anti-inflammatory drugs (NSAIDs) may have negative effects on fracture healing. This study aimed to assess the effect of immediate and delayed short-term administration of clinically relevant parecoxib doses and timing on fracture healing using an established animal fracture model. A standardized closed tibia shaft fracture was induced and stabilized by reamed intramedullary nailing in 66 Wistar rats. A ‘parecoxib immediate’ (Pi) group received parecoxib (3.2 mg/kg bodyweight twice per day) on days 0, 1, and 2. A ‘parecoxib delayed’ (Pd) group received the same dose of parecoxib on days 3, 4, and 5. A control group received saline only. Fracture healing was evaluated by biomechanical tests, histomorphometry, and dual-energy x-ray absorptiometry (DXA) at four weeks.Objectives
Methods
A balanced inflammatory response is important for successful fracture healing. The response of osteoporotic fracture healing is deranged and an altered inflammatory response can be one underlying cause. The objectives of this review were to compare the inflammatory responses between normal and osteoporotic fractures and to examine the potential effects on different healing outcomes. A systematic literature search was conducted with relevant keywords in PubMed, Embase, and Web of Science independently. Original preclinical studies and clinical studies involving the investigation of inflammatory response in fracture healing in ovariectomized (OVX) animals or osteoporotic/elderly patients with available full text and written in English were included. In total, 14 articles were selected. Various inflammatory factors were reported; of those tumour necrosis factor-α (TNF-α) and interleukin (IL)-6 are two commonly studied markers. Preclinical studies showed that OVX animals generally demonstrated higher systemic inflammatory response and poorer healing outcomes compared to normal controls (SHAM). However, it is inconclusive if the local inflammatory response is higher or lower in OVX animals. As for clinical studies, they mainly examine the temporal changes of the inflammatory stage or perform comparison between osteoporotic/fragility fracture patients and normal subjects without fracture. Our review of these studies emphasizes the lack of understanding that inflammation plays in the altered fracture healing response of osteoporotic/elderly patients. Taken together, it is clear that additional studies, preclinical and clinical, are required to dissect the regulatory role of inflammatory response in osteoporotic fracture healing. Cite this article:
The aim of this study was to systematically review the literature for evidence of the effect of a high-fat diet (HFD) on the onset or progression of osteoarthritis (OA) in mice. A literature search was performed in PubMed, Embase, Web of Science, and Scopus to find all studies on mice investigating the effects of HFD or Western-type diet on OA when compared with a control diet (CD). The primary outcome was the determination of cartilage loss and alteration. Secondary outcomes regarding local and systemic levels of proteins involved in inflammatory processes or cartilage metabolism were also examined when reported.Aims
Methods
The aims of this study were to determine whether the administration of anti-inflammatory and antifibrotic agents affect the proliferation, viability, and expression of markers involved in the fibrotic development of the fibroblasts obtained from arthrofibrotic tissue Dexamethasone, diclofenac, and decorin, in different concentrations, were employed to treat fibroblasts from arthrofibrotic tissue (AFib). Cell proliferation was measured by DNA quantitation, and viability was analyzed by Live/Dead staining. The levels of procollagen type I N-terminal propeptide (PINP) and procollagen type III N-terminal propeptide (PIIINP) were evaluated with enzyme-linked immunosorbent assay (ELISA) kits. In addition, the expressions of fibrotic markers were detected by real-time polymerase chain reaction (PCR). Fibroblasts isolated from healthy tissue (Fib) served as control. Further, a rabbit model of joint contracture was used to evaluate the antifibrotic effect of the three different agents.Objectives
Methods
Ligaments which heal spontaneously have a healing process that
is similar to skin wound healing. Menopause impairs skin wound healing
and may likewise impair ligament healing. Our purpose in this study
was to investigate the effect of surgical menopause on ligament
healing in a rabbit medial collateral ligament model. Surgical menopause was induced with ovariohysterectomy surgery
in adult female rabbits. Ligament injury was created by making a
surgical gap in the midsubstance of the medial collateral ligament.
Ligaments were allowed to heal for six or 14 weeks in the presence
or absence of oestrogen before being compared with uninjured ligaments. Molecular
assessment examined the messenger ribonucleic acid levels for collagens,
proteoglycans, proteinases, hormone receptors, growth factors and
inflammatory mediators. Mechanical assessments examined ligament
laxity, total creep strain and failure stress.Objectives
Methods
Emerging evidence indicates that tendon disease is an active process with inflammation that is critical to disease onset and progression. However, the key cytokines responsible for driving and sustaining inflammation have not been identified. We performed a systematic review of the literature using MEDLINE (U.S. National Library of Medicine, Bethesda, Maryland) in March 2017. Studies reporting the expression of interleukins (ILs), tumour necrosis factor alpha (TNF-α) and interferon gamma in diseased human tendon tissues, and animal models of tendon injury or exercise in comparison with healthy control tissues were included.Objectives
Methods
We have previously investigated an association between the genome copy number variation (CNV) and acetabular dysplasia (AD). Hip osteoarthritis is associated with a genetic polymorphism in the aspartic acid repeat in the N-terminal region of the asporin ( Acetabular coverage of all subjects was evaluated using radiological findings (Sharp angle, centre-edge (CE) angle, acetabular roof obliquity (ARO) angle, and minimum joint space width). Genomic DNA was extracted from peripheral blood leukocytes. Agilent’s region-targeted high-density oligonucleotide tiling microarray was used to analyse 64 female AD patients and 32 female control subjects. All statistical analyses were performed using EZR software (Fisher’s exact probability test, Pearson’s correlation test, and Student’s Objectives
Methods
Interleukin 18 (IL-18) is a regulatory cytokine that degrades the disc matrix. Bone morphogenetic protein-2 (BMP-2) stimulates synthesis of the disc extracellular matrix. However, the combined effects of BMP-2 and IL-18 on human intervertebral disc degeneration have not previously been reported. The aim of this study was to investigate the effects of the anabolic cytokine BMP-2 and the catabolic cytokine IL-18 on human nucleus pulposus (NP) and annulus fibrosus (AF) cells and, therefore, to identify potential therapeutic and clinical benefits of recombinant human (rh)BMP-2 in intervertebral disc degeneration. Levels of IL-18 were measured in the blood of patients with intervertebral disc degenerative disease and in control patients. Human NP and AF cells were cultured in a NP cell medium and treated with IL-18 or IL-18 plus BMP-2. mRNA levels of target genes were measured by real-time polymerase chain reaction, and protein levels of aggrecan, type II collagen, SOX6, and matrix metalloproteinase 13 (MMP13) were assessed by western blot analysis.Objectives
Methods
Sustained intra-articular delivery of pharmacological agents is an attractive modality but requires use of a safe carrier that would not induce cartilage damage or fibrosis. Collagen scaffolds are widely available and could be used intra-articularly, but no investigation has looked at the safety of collagen scaffolds within synovial joints. The aim of this study was to determine the safety of collagen scaffold implantation in a validated A total of 96 rabbits were randomly and equally assigned to four different groups: arthrotomy alone; arthrotomy and collagen scaffold placement; contracture surgery; and contracture surgery and collagen scaffold placement. Animals were killed in equal numbers at 72 hours, two weeks, eight weeks, and 24 weeks. Joint contracture was measured, and cartilage and synovial samples underwent histological analysis.Objectives
Materials and Methods
We aimed to assess the influence of ethnicity on the incidence
of heterotopic ossification (HO) after total hip arthroplasty (THA). We studied the six-month post-operative anteroposterior radiographs
of 1449 consecutive primary THAs (1324 patients) and retrospectively
graded them for the presence of HO, using the Brooker Classification. Aims
Patients and Methods
The aim of this study was to assess the effect
of injecting genetically engineered chondrocytes expressing transforming
growth factor beta 1 (TGF-β1) into the knees of patients with osteoarthritis.
We assessed the resultant function, pain and quality of life. A total of 54 patients (20 men, 34 women) who had a mean age
of 58 years (50 to 66) were blinded and randomised (1:1) to receive
a single injection of the active treatment or a placebo. We assessed
post-treatment function, pain severity, physical function, quality
of life and the incidence of treatment-associated adverse events. Patients
were followed at four, 12 and 24 weeks after injection. At final follow-up the treatment group had a significantly greater
improvement in the mean International Knee Documentation Committee
score than the placebo group (16 points; -18 to 49, This technique may result in improved clinical outcomes, with
the aim of slowing the degenerative process, leading to improvements
in pain and function. However, imaging and direct observational
studies are needed to verify cartilage regeneration. Nevertheless,
this study provided a sufficient basis to proceed to further clinical testing. Cite this article:
The most frequent cause of failure after total
hip replacement in all reported arthroplasty registries is peri-prosthetic
osteolysis. Osteolysis is an active biological process initiated
in response to wear debris. The eventual response to this process
is the activation of macrophages and loss of bone. Activation of macrophages initiates a complex biological cascade
resulting in the final common pathway of an increase in osteolytic
activity. The biological initiators, mechanisms for and regulation
of this process are beginning to be understood. This article explores current
concepts in the causes of, and underlying biological mechanism resulting
in peri-prosthetic osteolysis, reviewing the current basic science
and clinical literature surrounding the topic.
Surgical marking during tendon surgery is often used for technical
and teaching purposes. This study investigates the effect of a gentian
violet ink marker pen, a common surgical marker, on the viability
of the tissue and cells of tendon.
Objectives
Methods
The haematoma occurring at the site of a fracture is known to play an important role in bone healing. We have recently shown the presence of progenitor cells in human fracture haematoma and demonstrated that they have the capacity for multilineage mesenchymal differentiation. There have been many studies which have shown that low-intensity pulsed ultrasound (LIPUS) stimulates the differentiation of a variety of cells, but none has investigated the effects of LIPUS on cells derived from human fracture tissue including human fracture haematoma-derived progenitor cells (HCs). In this
Implantation of autologous chondrocytes and matrix autologous chondrocytes are techniques of cartilage repair used in the young adult knee which require harvesting of healthy cartilage and which may cause iatrogenic damage to the joint. This study explores alternative sources of autologous cells. Chondrocytes obtained from autologous bone-marrow-derived cells and those from the damaged cartilage within the lesion itself are shown to be viable alternatives to harvest-derived cells. A sufficient number and quality of cells were obtained by the new techniques and may be suitable for autologous chondrocyte and matrix autologous chondrocyte implantation.
We used a canine intercalary bone defect model to determine the effects of recombinant human osteogenic protein 1 (rhOP-1) on allograft incorporation. The allograft was treated with an implant made up of rhOP-1 and type I collagen or with type I collagen alone. Radiographic analysis showed an increased volume of periosteal callus in both test groups compared with the control group at weeks 4, 6, 8 and 10. Mechanical testing after 12 weeks revealed increased maximal torque and stiffness in the rhOP-1 treated groups compared with the control group. These results indicate a benefit from the use of an rhOP-1 implant in the healing of bone allografts. The effect was independent of the position of the implant. There may be a beneficial clinical application for this treatment.
Articular cartilage repair remains a challenge to surgeons and basic scientists. The field of tissue engineering allows the simultaneous use of material scaffolds, cells and signalling molecules to attempt to modulate the regenerative tissue. This review summarises the research that has been undertaken to date using this approach, with a particular emphasis on those techniques that have been introduced into clinical practice, via in vitro and preclinical studies.
