Inflammatory Markers Involved in the Pathogenesis of Dupuytren's Contracture.

Dupuytren's disease is a common fibroproliferative disease that can result in debilitating hand deformities. Partial correction and return of deformity are common with surgical or clinical treatments at present. While current treatments are limited to local procedures for relatively late effects of the disease, the pathophysiology of this connective tissue disorder is associated with both local and systemic processes (e.g., fibrosis, inflammation). Hence, a better understanding of the systemic circulation of Dupuytren related cytokines and growth factors may provide important insights into disease progression. In addition, systemic biomarker analysis could yield new concepts for treatments of Dupuytren that attenuate circulatory factors (e.g., anti-inflammatory agents, neutralizing antibodies). Progress in the development of any disease modifying biologic treatment for Dupuytren has been hampered by the lack of clinically useful biomarkers. The characterization of nonsurgical Dupuytren biomarkers will permit disease staging from diagnostic and prognostic perspectives, as well as allows evaluation of biologic responses to treatment. Identification of such markers may transcend their use in Dupuytren treatment, because fibrotic biological processes fundamental to Dupuytren are relevant to fibrosis in many other connective tissues and organs with collagen-based tissue compartments. There is a wide range of potential Dupuytren biomarker categories that could be informative, including disease determinants linked to genetics, collagen metabolism, as well as immunity and inflammation (e.g., cytokines, chemokines). This narrative review provides a broad overview of previous studies and emphasizes the importance of inflammatory mediators as candidate circulating biomarkers for monitoring Dupuytren's disease.

DNA Double-Strand Break Repair Kinetics after Exposure to Photons and Ions: A Systematic Review.

This study offers a review of published data on DNA double strand break (DSB) repair kinetics after exposure to ionizing radiation. By compiling a database, which currently includes 285 DNA DSB repair experiments utilizing both photons and ions, we investigate the impact of distinct experimental parameters on the kinetics of DNA DSB repair. Methodological differences and inconsistencies in reporting make the comparison of data generated by different research groups challenging. Nevertheless, by implementing filtering criteria, we can compare repair kinetics obtained with normal and tumor cells derived from human or animal tissues, as well as cells exposed to photons or ions ranging from hydrogen to iron ions. In addition, several repair curves of repair deficient cell lines were included. The study aims to provide researchers with a comprehensive overview of experimental factors that may confound results and emphasize the importance of precise reporting of experimental parameters. Moreover, we identify gaps in the literature that require attention in future studies, aiming to address clinically relevant questions related to radiotherapy. The database can be freely accessed at: https://github.com/weradstake/DRDNA.

Characterizing Proton-Induced Biological Effects in a Mouse Spinal Cord Model: A Comparison of Bragg Peak and Entrance Beam Response in Single and Fractionated Exposures.

PURPOSE: Proton relative biological effectiveness (RBE) is a dynamic variable influenced by factors like linear energy transfer (LET), dose, tissue type, and biological endpoint. The standard fixed proton RBE of 1.1, currently used in clinical planning, may not accurately represent the true biological effects of proton therapy (PT) in all cases. This uncertainty can contribute to radiation-induced normal tissue toxicity in patients. In late-responding tissues such as the spinal cord, toxicity can cause devastating complications. This study investigated spinal cord tolerance in mice subjected to proton irradiation and characterized the influence of fractionation on proton- induced myelopathy at entrance (ENT) and Bragg peak (BP) positions. METHODS AND MATERIALS: Cervical spinal cords of 8-week-old C57BL/6J female mice were irradiated with single- or multi-fractions (18x) using lateral opposed radiation fields at 1 of 2 positions along the Bragg curve: ENT (dose-mean LET = 1.2 keV/µm) and BP (LET = 6.9 keV/µm). Mice were monitored over 1 year for changes in weight, mobility, and general health, with radiation-induced myelopathy as the primary biological endpoint. Calculations of the RBE of the ENT and BP curve (RBEENT/BP) were performed. RESULTS: Single-fraction RBEENT/BP for 50% effect probability (tolerance dose (TD50), grade II paresis, determined using log-logistic model fitting) was 1.10 ± 0.06 (95% CI) and for multifraction treatments it was 1.19 ± 0.05 (95% CI). Higher incidence and faster onset of paralysis were seen in mice treated at the BP compared with ENT. CONCLUSIONS: The findings challenge the universally fixed RBE value in PT, indicating up to a 25% mouse spinal cord RBEENT/BP variation for multifraction treatments. These results highlight the importance of considering fractionation in determining RBE for PT. Robust characterization of proton-induced toxicity, aided by in vivo models, is paramount for refining clinical decision-making and mitigating potential patient side effects.

BMP2 and GDF5 for Compartmentalized Regeneration of the Scapholunate Ligament.

Background Chronic injuries to the scapholunate ligament (SLIL) alter carpal kinematics and may progress to early degenerative osteoarthritis. To date, there is no consensus for the best method for SLIL reconstruction. This study aims to assess the use of growth factors (bone morphogenetic protein [BMP]2 and growth and differentiation factor 5 [GDF5]) for compartmentalized regeneration of bone and ligament in this multiphasic scaffold in a rabbit knee model. Case Description A total of 100 µg of BMP2 and 30 µg of GDF5 were encapsulated into a heparinized gelatin-hyaluronic acid hydrogel and loaded into the appropriate compartment of the multiphasic scaffold. The multiphasic scaffold was implanted to replace the native rabbit medial collateral ligament ( n = 16). The rabbits were randomly assigned to two different treatment groups. The first group was immobilized postoperatively with the knee pinned in flexion with K-wires for 4 weeks ( n = 8) prior to sacrifice. The second group was immobilized for 4 weeks, had the K-wires removed followed by a further 4 weeks of mobilization prior to sample harvesting. Literature Review Heterotopic ossification as early as 4 weeks was noted on gross dissection and confirmed by microcomputed tomography and histological staining. This analysis revealed formation of a bony bridge located within and over the ligament compartment in the intra-articular region. Biomechanical testing showed increased ultimate force of the ligament compartment at 4 weeks postimplantation consistent with the presence of bone formation and higher numbers of scaffold failures at the bone-tendon junction. This study has demonstrated that the addition of BMP2 and GDF5 in the bone-ligament-bone (BLB) scaffold resulted in heterotopic bone formation and failure of the ligament compartment. Clinical Relevance The implantation of a three-dimensional-printed BLB scaffold alone demonstrated superior biomechanical and histological results, and further investigation is needed as a possible clinical reconstruction for the SLIL.

Vitamin C epigenetically controls osteogenesis and bone mineralization.

Vitamin C deficiency disrupts the integrity of connective tissues including bone. For decades this function has been primarily attributed to Vitamin C as a cofactor for collagen maturation. Here, we demonstrate that Vitamin C epigenetically orchestrates osteogenic differentiation and function by modulating chromatin accessibility and priming transcriptional activity. Vitamin C regulates histone demethylation (H3K9me3 and H3K27me3) and promotes TET-mediated 5hmC DNA hydroxymethylation at promoters, enhancers and super-enhancers near bone-specific genes. This epigenetic circuit licenses osteoblastogenesis by permitting the expression of all major pro-osteogenic genes. Osteogenic cell differentiation is strictly and continuously dependent on Vitamin C, whereas Vitamin C is dispensable for adipogenesis. Importantly, deletion of 5hmC-writers, Tet1 and Tet2, in Vitamin C-sufficient murine bone causes severe skeletal defects which mimic bone phenotypes of Vitamin C-insufficient Gulo knockout mice, a model of Vitamin C deficiency and scurvy. Thus, Vitamin C's epigenetic functions are central to osteoblastogenesis and bone formation and may be leveraged to prevent common bone-degenerating conditions.

MicroRNA-101a enhances trabecular bone accrual in male mice.

High-throughput microRNA sequencing was performed during differentiation of MC3T3-E1 osteoblasts to develop working hypotheses for specific microRNAs that control osteogenesis. The expression data show that miR-101a, which targets the mRNAs for the epigenetic enzyme Ezh2 and many other proteins, is highly upregulated during osteoblast differentiation and robustly expressed in mouse calvaria. Transient elevation of miR-101a suppresses Ezh2 levels, reduces tri-methylation of lysine 27 in histone 3 (H3K27me3; a heterochromatic mark catalyzed by Ezh2), and accelerates mineralization of MC3T3-E1 osteoblasts. We also examined skeletal phenotypes of an inducible miR-101a transgene under direct control of doxycycline administration. Experimental controls and mir-101a over-expressing mice were exposed to doxycycline in utero and postnatally (up to 8 weeks of age) to maximize penetrance of skeletal phenotypes. Male mice that over-express miR-101a have increased total body weight and longer femora. MicroCT analysis indicate that these mice have increased trabecular bone volume fraction, trabecular number and trabecular thickness with reduced trabecular spacing as compared to controls. Histomorphometric analysis demonstrates a significant reduction in osteoid volume to bone volume and osteoid surface to bone surface. Remarkably, while female mice also exhibit a significant increase in bone length, no significant changes were noted by microCT (trabecular bone parameters) and histomorphometry (osteoid parameters). Hence, miR-101a upregulation during osteoblast maturation and the concomitant reduction in Ezh2 mediated H3K27me3 levels may contribute to the enhanced trabecular bone parameters in male mice. However, the sex-specific effect of miR-101a indicates that more intricate epigenetic mechanisms mediate physiological control of bone formation and homeostasis.

Surface Roughness of Titanium Orthopedic Implants Alters the Biological Phenotype of Human Mesenchymal Stromal Cells.

Metal orthopedic implants are largely biocompatible and generally achieve long-term structural fixation. However, some orthopedic implants may loosen over time even in the absence of infection. In vivo fixation failure is multifactorial, but the fundamental biological defect is cellular dysfunction at the host-implant interface. Strategies to reduce the risk of short- and long-term loosening include surface modifications, implant metal alloy type, and adjuvant substances such as polymethylmethacrylate cement. Surface modifications (e.g., increased surface rugosity) can increase osseointegration and biological ingrowth of orthopedic implants. However, the localized responses of cells to implant surface modifications need to be better characterized. As an in vitro model for investigating cellular responses to metallic orthopedic implants, we cultured mesenchymal stromal/stem cells on clinical-grade titanium disks (Ti6Al4V) that differed in surface roughness as high (porous structured), medium (grit blasted), and low (bead blasted). Topological characterization of clinically relevant titanium (Ti) materials combined with differential mRNA expression analyses (RNA-seq and real-time quantitative polymerase chain reaction) revealed alterations to the biological phenotype of cells cultured on titanium structures that favor early extracellular matrix production and observable responses to oxidative stress and heavy metal stress. These results provide a descriptive model for the interpretation of cellular responses at the interface between native host tissues and three-dimensionally printed modular orthopedic implants, and will guide future studies aimed at increasing the long-term retention of such materials after total joint arthroplasty. Impact statement Using an in vitro model of implant-to-cell interactions by culturing mesenchymal stromal cells (MSCs) on clinically relevant titanium materials of varying topological roughness, we identified mRNA expression patterns consistent with early extracellular matrix (ECM) production and responses to oxidative/heavy metal stress. Implants with high surface roughness may delay the differentiation and ECM formation of MSCs and alter the expression of genes sensitive to reactive oxygen species and protein kinases. In combination with ongoing animal studies, these results will guide future studies aimed at increasing the long-term retention of widely used titanium materials after total joint arthroplasty.

Dosimetric Assessment of a High Precision System for Mouse Proton Irradiation to Assess Spinal Cord Toxicity.

The uncertainty associated with the relative biological effectiveness (RBE) in proton therapy, particularly near the Bragg peak (BP), has led to the shift towards biological-based treatment planning. Proton RBE uncertainty has recently been reported as a possible cause for brainstem necrosis in pediatric patients treated with proton therapy. Despite this, in vivo studies have been limited due to the complexity of accurate delivery and absolute dosimetry. The purpose of this investigation was to create a precise and efficient method of treating the mouse spinal cord with various portions of the proton Bragg curve and to quantify associated uncertainties for the characterization of proton RBE. Mice were restrained in 3D printed acrylic boxes, shaped to their external contour, with a silicone insert extending down to mold around the mouse. Brass collimators were designed for parallel opposed beams to treat the spinal cord while shielding the brain and upper extremities of the animal. Up to six animals may be accommodated for simultaneous treatment within the restraint system. Two plans were generated targeting the cervical spinal cord, with either the entrance (ENT) or the BP portion of the beam. Dosimetric uncertainty was measured using EBT3 radiochromic film with a dose-averaged linear energy transfer (LETd) correction. Positional uncertainty was assessed by collecting a library of live mouse scans (n = 6 mice, two independent scans per mouse) and comparing the following dosimetric statistics from the mouse cervical spinal cord: Volume receiving 90% of the prescription dose (V90); mean dose to the spinal cord; and LETd. Film analysis results showed the dosimetric uncertainty to be ±1.2% and ±5.4% for the ENT and BP plans, respectively. Preliminary results from the mouse library showed the V90 to be 96.3 ± 4.8% for the BP plan. Positional uncertainty of the ENT plan was not measured due to the inherent robustness of that treatment plan. The proposed high-throughput mouse proton irradiation setup resulted in accurate dose delivery to mouse spinal cords positioned along the ENT and BP. Future directions include adapting the setup to account for weight fluctuations in mice undergoing fractionated irradiation.

Inhibition of ATM Induces Hypersensitivity to Proton Irradiation by Upregulating Toxic End Joining.

Proton Bragg peak irradiation has a higher ionizing density than conventional photon irradiation or the entrance of the proton beam profile. Whether targeting the DNA damage response (DDR) could enhance vulnerability to the distinct pattern of damage induced by proton Bragg peak irradiation is currently unknown. Here, we performed genetic or pharmacologic manipulation of key DDR elements and evaluated DNA damage signaling, DNA repair, and tumor control in cell lines and xenografts treated with the same physical dose across a radiotherapy linear energy transfer spectrum. Radiotherapy consisted of 6 MV photons and the entrance beam or Bragg peak of a 76.8 MeV spot scanning proton beam. More complex DNA double-strand breaks (DSB) induced by Bragg peak proton irradiation preferentially underwent resection and engaged homologous recombination (HR) machinery. Unexpectedly, the ataxia-telangiectasia mutated (ATM) inhibitor, AZD0156, but not an inhibitor of ATM and Rad3-related, rendered cells hypersensitive to more densely ionizing proton Bragg peak irradiation. ATM inhibition blocked resection and shunted more DSBs to processing by toxic ligation through nonhomologous end-joining, whereas loss of DNA ligation via XRCC4 or Lig4 knockdown rescued resection and abolished the enhanced Bragg peak cell killing. Proton Bragg peak monotherapy selectively sensitized cell lines and tumor xenografts with inherent HR defects, and the repair defect induced by ATM inhibitor coadministration showed enhanced efficacy in HR-proficient models. In summary, inherent defects in HR or administration of an ATM inhibitor in HR-proficient tumors selectively enhances the relative biological effectiveness of proton Bragg peak irradiation. SIGNIFICANCE: Coadministration of an ATM inhibitor rewires DNA repair machinery to render cancer cells uniquely hypersensitive to DNA damage induced by the proton Bragg peak, which is characterized by higher density ionization.See related commentary by Nickoloff, p. 3156.

Modernizing Storage Conditions for Fresh Osteochondral Allografts by Optimizing Viability at Physiologic Temperatures and Conditions.

Objective. Osteochondral allograft (OCA) transplantation has demonstrated good long-term outcomes in treatment of cartilage defects. Viability, a key factor in clinical success, decreases with peri-implantation storage at 4°C during pathogen testing, matching logistics, and transportation. Modern, physiologic storage conditions may improve viability and enhance outcomes. Design. Osteochondral specimens from total knee arthroplasty patients (6 males, 5 females, age 56.4 ± 2.2 years) were stored in media and incubated at normoxia (21% O2) at 22°C or 37°C, and hypoxia (2% O2) at 37°C. Histology, live-dead staining, and quantitative polymerase chain reaction (qPCR) was performed 24 hours after harvest and following 7 days of incubation. Tissue architecture, cell viability, and gene expression were analyzed. Results. No significant viability or gene expression deterioration of cartilage was observed 1-week postincubation at 37°C, with or without hypoxia. Baseline viable cell density (VCD) was 94.0% ± 2.7% at day 1. At day 7, VCD was 95.1% (37°C) with normoxic storage and 92.2% (37°C) with hypoxic storage (P ≥ 0.27). Day 7 VCD (22°C) incubation was significantly lower than both the baseline and 37°C storage values (65.6%; P < 0.01). COL1A1, COL1A2, and ACAN qPCR expression was unchanged from baseline (P < 0.05) for all storage conditions at day 7, while CD163 expression, indicative of inflammatory macrophages and monocytes, was significantly lower in the 37°C groups (P < 0.01). Conclusion. Physiologic storage at 37°C demonstrates improved chondrocyte viability and metabolism, and maintained collagen expression compared with storage at 22°C. These novel findings guide development of a method to optimize short-term fresh OCA storage, which may lead to improved clinical results.

Fresh Osteochondral Allograft Transplantation in the Knee: A Viability and Histologic Analysis for Optimizing Graft Viability and Expanding Existing Standard Processed Graft Resources Using a Living Donor Cartilage Program.

OBJECTIVE: This study aims to (1) determine and validate living cartilage allograft transplantation as a novel source for viable osteochondral allograft (OCA) tissues and (2) perform histologic and viability comparisons of living donor cartilage tissues to currently available clinical-grade standard processed grafts. DESIGN: Using healthy cartilage from well-preserved contralateral compartments in 27 patients undergoing total knee arthroplasty (TKA) and 10 clinical-grade OCA specimens obtained immediately following operative implantation, standard and living donor OCA quality was evaluated at the time of harvest and following up to 3 weeks of storage on the basis of macroscopic International Cartilage Repair Society grade, histology, and viability. RESULTS: Osteochondral samples demonstrated a consistent decrease in viability and histologic quality over the first 3 weeks of storage at 37°C, supporting the utility of an OCA paradigm shift toward early implantation, as was the clinical standard up until recent adoption of transplantation at 14 to 35 days following donor procurement. Samples from the 10 clinical-grade OCAs, implanted at an average of 23 days following graft harvest demonstrated a mean viable cell density of 45.6% at implantation, significantly lower (P < 0.01) than the 93.6% viability observed in living donor allograft tissues. CONCLUSIONS: Osteochondral tissue viability and histologic quality progressively decreases with ex vivo storage, even when kept at physiologic temperatures. Currently available clinical OCAs are stored for 2 to 5 weeks prior to implantation and demonstrate inferior viability to that of fresh osteochondral tissues that can be made available through the use of a living donor cartilage program.

Multiphasic scaffold for scapholunate interosseous ligament reconstruction: A study in the rabbit knee.

Scapholunate interosseous ligament tears are a common wrist injury in young and active patients that can lead to suboptimal outcomes after repair. This research aims to assess a multiphasic scaffold using 3D-printing for reconstruction of the dorsal scapholunate interosseous ligament. The scaffold was surgically implanted in vivo in the position of the native rabbit medial collateral ligament. Two branches of treatment were implemented in the study. In the first group, the rabbits (n = 8) had the knee joint fixed in flexion for 4 weeks using 1.4 mm K-wires prior to sample harvesting. The second group (n = 8) had the rabbit knee joint immobilized for 4 weeks prior to K-wire removal and mobilization for an additional 4 weeks prior to sample harvesting. Overall, samples were harvested at 4 weeks post-surgery (immobilized group) and eight weeks post-surgery (mobilized group). Mechanical tensile testing (n = 5/group) and histology (n = 3/group) of the constructs were conducted. Tissue integration and maturation were observed resulting in increased mechanical strength of the operated joint at 8 weeks (P < .05). Bone and ligament tissues were regenerated in their respective compartments with structural and mechanical properties approaching those reported for the human dorsal SLIL ligament. Clinical Significance: This proof of concept study has demonstrated that the synthetic multiphasic scaffold was capable of regenerating both bone and ligament while also withstanding the physiological load once implanted in the rabbit knee. The artificial scaffold may provide an alternative to current techniques for reconstruction of scapholunate instability or other ligament injuries in the hand and wrist.

A High-Precision Method for In Vitro Proton Irradiation.

PURPOSE: Although proton therapy has become a well-established radiation modality, continued efforts are needed to improve our understanding of the molecular and cellular mechanisms occurring during treatment. Such studies are challenging, requiring many resources. The purpose of this study was to create a phantom that would allow multiple in vitro experiments to be irradiated simultaneously with a spot-scanning proton beam. MATERIALS AND METHODS: The setup included a modified patient-couch top coupled with a high-precision robotic arm for positioning. An acrylic phantom was created to hold 4 6-well cell-culture plates at 2 different positions along the Bragg curve in a reproducible manner. The proton treatment plan consisted of 1 large field encompassing all 4 plates with a monoenergetic 76.8-MeV posterior beam. For robust delivery, a mini pyramid filter was used to broaden the Bragg peak (BP) in the depth direction. Both a Markus ionization chamber and EBT3 radiochromic film measurements were used to verify absolute dose. RESULTS: A treatment plan for the simultaneous irradiation of 2 plates irradiated with high linear energy transfer protons (BP, 7 keV/µm) and 2 plates irradiated with low linear energy transfer protons (entrance, 2.2 keV/µm) was created. Dose uncertainty was larger across the setup for cell plates positioned at the BP because of beam divergence and, subsequently, variable proton-path lengths. Markus chamber measurements resulted in uncertainty values of ±1.8% from the mean dose. Negligible differences were seen in the entrance region (<0.3%). CONCLUSION: The proposed proton irradiation setup allows 4 plates to be simultaneously irradiated with 2 different portions (entrance and BP) of a 76.8-MeV beam. Dosimetric uncertainties across the setup are within ±1.8% of the mean dose.

A multi-chamber tissue culture device for load-dependent parallel evaluation of tendon explants.

BACKGROUND: Injuries in the musculoskeletal system, such as tendon and ligament ruptures, are challenging to manage and often require surgical reconstructions with limited long-term success. Thus, characterizations of these tissues are urgently needed to better understand cellular mechanisms that regulate tissue homeostasis and healing. Explant culturing systems allow for ex vivo analysis of tissues in an environment that mimics the native microenvironment in vivo. METHODS: Collaborative efforts within our institution facilitated the establishment of a novel explant culturing system. Tissue specimens cultured in single wells, with individual applied loading and/or biological environment, allowed characterization of tissue cultured under a variety of biological loading conditions. Quantitative PCR analysis for selected gene markers was our primary outcome. RESULTS: Data were stratified for analysis by either culture environment or loading condition. Our gene expression results show that specimens clustered by culture condition may differ in molecular markers related to ECM production (e.g., Col1a1, Adamts4) and/or organization (e.g., Tnc, Dnc). In contrast, loading condition did significantly alter the median gene expression levels of tissues in comparison to unloaded control samples, although gene expression values related to ECM degradation (e.g., Mmp1, Mmp10) were altered in tendons cultured under tension in the device. CONCLUSION: Our study demonstrates promising utility of a novel explant culturing system for further characterization of musculoskeletal tissues such as native tendons and ligaments, as well as pathologic fibrotic tissues resulting from arthrofibrosis or Dupuytren's disease.

In vivo assessment of high-molecular-weight polyethylene core suture tape for intra-articular ligament reconstruction: an animal study.

AIMS: Options for the treatment of intra-articular ligament injuries are limited, and insufficient ligament reconstruction can cause painful joint instability, loss of function, and progressive development of degenerative arthritis. This study aimed to assess the capability of a biologically enhanced matrix material for ligament reconstruction to withstand tensile forces within the joint and enhance ligament regeneration needed to regain joint function. MATERIALS AND METHODS: A total of 18 New Zealand rabbits underwent bilateral anterior cruciate ligament reconstruction by autograft, FiberTape, or FiberTape-augmented autograft. Primary outcomes were biomechanical assessment (n = 17), microCT (µCT) assessment (n = 12), histological evaluation (n = 12), and quantitative polymerase chain reaction (qPCR) analysis (n = 6). RESULTS: At eight weeks, FiberTape alone or FiberTape-augmented autograft demonstrated increased biomechanical stability compared with autograft regarding ultimate load to failure (p = 0.035), elongation (p = 0.006), and energy absorption (p = 0.022). FiberTape-grafted samples also demonstrated increased bone mineral density in the bone tunnel (p = 0.039). Histological evaluation showed integration of all grafts in the bone tunnels by new bone formation, and limited signs of inflammation overall. A lack of prolonged inflammation in all samples was confirmed by quantification of inflammation biomarkers. However, no regeneration of ligament-like tissue was observed along the suture tape materials. Except for one autograft failure, no adverse events were detected. CONCLUSION: Our results indicate that FiberTape increases the biomechanical performance of intra-articular ligament reconstructions in a verified rabbit model at eight weeks. Within this period, FiberTape did not adversely affect bone tunnel healing or invoke a prolonged elevation in inflammation. Cite this article: Bone Joint J 2019;101-B:1238-1247.

Effect of Lidocaine on Viability and Gene Expression of Human Adipose-derived Mesenchymal Stem Cells: An in vitro Study.

OBJECTIVE: To assess the biologic effects of lidocaine on the viability, proliferation, and function of human adipose tissue-derived mesenchymal stromal/stem cells (MSCs) in vitro. METHODS: Adipose-derived MSCs from three donors were exposed to lidocaine at various dilutions (2 mg/mL to 8 mg/mL) and exposure times (0.5 to 4 hours). Cell number and viability, mitochondrial activity, and real-time reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) were analyzed at 0 (immediate effects) or 24 and 48 hours (recovery effects) after treatment with lidocaine. RESULTS: Trypan blue staining showed that increasing concentrations of lidocaine decreased the number of observable viable cells. 3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium (MTS) assays revealed a concentration- and time- dependent decline of mitochondrial activity and proliferative ability. Gene expression analysis by RT-qPCR revealed that adipose-derived MSCs exposed to lidocaine express robust levels of stress response/cytoprotective genes. However, higher concentrations of lidocaine caused a significant downregulation of these genes. No significant differences were observed in expression of extracellular matrix (ECM) markers COL1A1 and DCN except for COL3A1 (P < .05). Levels of messenger RNA (mRNA) for proliferation markers (CCNB2, HIST2H4A, P < .001) and MKI67 (P < .001) increased at 24 and 48 hours. Expression levels of several transcription factors- including SP1, PRRX1, and ATF1-were modulated in the same manner. MSC surface markers CD44 and CD105 demonstrated decreased expression immediately after treatment, but at 24 and 48 hours postexposure, the MSC markers showed no significant difference among groups. CONCLUSION: Lidocaine is toxic to MSCs in a dose- and time- dependent manner. MSC exposure to high (4-8 mg/mL) concentrations of lidocaine for prolonged periods can affect their biologic functions. Although the exposure time in vivo is short, it is essential to choose safe concentrations when applying lidocaine along with MSCs to avoid compromising the viability and potency of the stem cell therapy.

Extracellular matrix protein production in human adipose-derived mesenchymal stem cells on three-dimensional polycaprolactone (PCL) scaffolds responds to GDF5 or FGF2.

PURPOSE: The poor healing potential of intra-articular ligament injuries drives a need for the development of novel, viable 'neo-ligament' alternatives. Ex vivo approaches combining stem cell engineering, 3-dimensional biocompatible scaffold design and enhancement of biological and biomechanical functionality via the introduction of key growth factors and morphogens, represent a promising solution to ligament regeneration. METHODS: We investigated growth, differentiation and extracellular matrix (ECM) protein production of human adipose-derived mesenchymal stem/stromal cells (MSCs), cultured in 5% human platelet lysate (PL) and seeded on three-dimensional polycaprolactone (PCL) scaffolds, in response to the connective-tissue related ligands fibroblast growth factor 2 (basic) (FGF2) and growth and differentiation factor-5 (GDF5). Phenotypic alterations of MSCs under different biological conditions were examined using cell viability assays, real time qPCR analysis of total RNA, as well as immunofluorescence microscopy. RESULTS: Phenotypic conversion of MSCs into ECM producing fibroblastic cells proceeds spontaneously in the presence of human platelet lysate. Administration of FGF2 and/or GDF5 enhances production of mRNAs for several ECM proteins including Collagen types I and III, as well as Tenomodulin (e.g., COL1A1, TNMD), but not Tenascin-C (TNC). Differences in the in situ deposition of ECM proteins Collagen type III and Tenascin-C were validated by immunofluorescence microscopy. SUMMARY: Treatment of MSCs with FGF2 and GDF5 was not synergistic and occasionally antagonistic for ECM production. Our results suggest that GDF5 alone enhances the conversion of MSCs to fibroblastic cells possessing a phenotype consistent with that of connective-tissue fibroblasts.

Fibrin glue mediated delivery of bone anabolic reagents to enhance healing of tendon to bone.

Tendon graft healing in bone tunnels for the fixation of intra-articular ligament reconstructions may limit clinical outcome by delaying healing. This study assesses the effects of hydrogel-mediated delivery of bone anabolic growth factors in a validated model of tendon-to-bone tunnel healing. Forty-five Wistar rats were randomly allocated into three groups (BMP2-treated, GSK126-treated, and placebo). All animals underwent a tendon-to-bone tunnel reconstruction. Healing was evaluated at 4 weeks by biomechanical assessment, micro-computed tomography (bone mineral density, bone volume, cross sectional area of bone tunnels), and traditional histology. Adverse events associated with the hydrogel-mediated delivery of drugs were not observed. Results of our biomechanical assessment demonstrated favorable trends in animals treated with bone anabolic factors for energy absorption (P = 0.116) and elongation (P = 0.054), while results for force to failure (P = 0.691) and stiffness (P = 0.404) did not show discernible differences. Cross sectional areas for BMP2-treated animals were reduced, but neither BMP2 nor GSK126 administration altered bone mineral density (P = 0.492) or bone volume in the bone tunnel. These results suggest a novel and positive effect of bone anabolic factors on tendon-to-bone tunnel healing. Histological evaluation confirmed absence of collagen fibers crossing the soft tissue-bone interface indicating immature graft integration as expected at this time point. Our study indicates that hydrogel-mediated delivery of BMP2 and GSK126 appears to be safe and has the potential to enhance tendon-to-bone tunnel healing in ligament reconstructions.

Safety Studies for Use of Adipose Tissue-Derived Mesenchymal Stromal/Stem Cells in a Rabbit Model for Osteoarthritis to Support a Phase I Clinical Trial.

Adipose-derived mesenchymal stem cells (AMSCs) offer potential as a therapeutic option for clinical applications in musculoskeletal regenerative medicine because of their immunomodulatory functions and capacity for trilineage differentiation. In preparation for a phase I clinical trial using AMSCs to treat patients with osteoarthritis, we carried out preclinical studies to assess the safety of human AMSCs within the intra-articular joint space. Culture-expanded human AMSCs grown in human platelet-lysate were delivered via intra-articular injections into normal healthy rabbit knees and knees at risk for the development of osteoarthritis after bilateral medial anterior hemimeniscectomy. Treatment outcomes and safety were evaluated by assessing the general health, function, and behavior of the animals. Joint tissues were analyzed by x-ray, magnetic resonance imaging, and histopathology. Intra-articular AMSC therapy was well tolerated in this study. We did not observe adverse systemic reactions, nor did we find evidence of damage to intra-articular joint tissues. Thus, the data generated in this study show a favorable safety profile for AMSCs within the joint space in support of a phase I clinical trial evaluating the clinical utility of AMSCs to treat osteoarthritis. Stem Cells Translational Medicine 2017;6:910-922.

Contrast imaging in mouse embryos using high-frequency ultrasound.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or αvß3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.