Moderate and intensive mechanical loading differentially modulate the phenotype of tendon stem/progenitor cells in vivo

To examine the differential mechanobiological responses of specific resident tendon cells, we developed an in vivo model of whole-body irradiation followed by injection of either tendon stem/progenitor cells (TSCs) expressing green fluorescent protein (GFP-TSCs) or mature tenocytes expressing GFP (GFP-TNCs) into the patellar tendons of wild type C57 mice. Injected mice were subjected to short term (3 weeks) treadmill running, specifically moderate treadmill running (MTR) and intensive treadmill running (ITR). In MTR mice, both GFP-TSC and GFP-TNC injected tendons maintained normal cell morphology with elevated expression of tendon related markers collagen I and tenomodulin. In ITR mice injected with GFP-TNCs, cells also maintained an elongated shape similar to the shape found in normal/ untreated control mice, as well as elevated expression of tendon related markers. However, ITR mice injected with GFP-TSCs showed abnormal changes, such as cell morphology transitioning to a round shape, elevated chondrogenic differentiation, and increased gene expression of non-tenocyte related genes LPL, Runx-2, and SOX-9. Increased gene expression data was supported by immunostaining showing elevated expression of SOX-9, Runx-2, and PPARγ. This study provides evidence that while MTR maintains tendon homeostasis by promoting the differentiation of TSCs into TNCs, ITR causes the onset of tendinopathy development by inducing non-tenocyte differentiation of TSCs, which may eventually lead to the formation of non-tendinous tissues in tendon tissue after long term mechanical overloading conditions on the tendon

Moderate and intensive mechanical loading differentially modulate the phenotype of tendon stem/progenitor cells in vivo.

To examine the differential mechanobiological responses of specific resident tendon cells, we developed an in vivo model of whole-body irradiation followed by injection of either tendon stem/progenitor cells (TSCs) expressing green fluorescent protein (GFP-TSCs) or mature tenocytes expressing GFP (GFP-TNCs) into the patellar tendons of wild type C57 mice. Injected mice were subjected to short term (3 weeks) treadmill running, specifically moderate treadmill running (MTR) and intensive treadmill running (ITR). In MTR mice, both GFP-TSC and GFP-TNC injected tendons maintained normal cell morphology with elevated expression of tendon related markers collagen I and tenomodulin. In ITR mice injected with GFP-TNCs, cells also maintained an elongated shape similar to the shape found in normal/untreated control mice, as well as elevated expression of tendon related markers. However, ITR mice injected with GFP-TSCs showed abnormal changes, such as cell morphology transitioning to a round shape, elevated chondrogenic differentiation, and increased gene expression of non-tenocyte related genes LPL, Runx-2, and SOX-9. Increased gene expression data was supported by immunostaining showing elevated expression of SOX-9, Runx-2, and PPARγ. This study provides evidence that while MTR maintains tendon homeostasis by promoting the differentiation of TSCs into TNCs, ITR causes the onset of tendinopathy development by inducing non-tenocyte differentiation of TSCs, which may eventually lead to the formation of non-tendinous tissues in tendon tissue after long term mechanical overloading conditions on the tendon

Fully Reduced HMGB1 Enhances Nonunion Fracture Healing in Diabetic Rat Model

Category: Basic Sciences/Biologics; Diabetes Introduction/Purpose: Diabetes mellitus is a chronic metabolic disease that leads to serious complications including bone health and wound healing. Bone fractures commonly occur in diabetic patients, and are difficultly to heal due to prolonged inflammation, which leads to a higher risk of non-union. Fully reduced high mobility group box1 (frHMGB1) can play an important role in skeletal regeneration; however, the activity can be inhibited by Metformin; which is commonly used for the treatment of diabetes. The effect of frHMGB1 and Metformin on the bone fracture healing is largely unknown. In this study, we tested the hypothesis that frHMGB1 enhances healing in a nonunion bone fracture and Metformin inhibits the healing using a diabetic rat model. Methods: Diabetic fracture rat model was created by injection of streptozotocin into total 32 Sprague Dawley rats and confirmed by blood glucose levels.An incision was performed, tibia bones were fractured, followed by the muscle and skin wound closure.The rats were divided into four groups with 8 rats per group(Fig 1): Group-1 had no additional treatment;Group-2 were injected Metformin (IP, 160 mg/kg) daily;Group-3 were injected frHMGB1 into wound area (250 µg/kg) weekly;Group-4 were injected Metformin (IP, 160 mg/kg) daily and frHMGB1 into wound area (250 µg/kg) weekly. The rats were monitored postoperatively and sacrificed on postoperative day-28 and day-90. The blood samples were collected for HMGB1 & IL-1β.Tibia samples were also collected and examined by gross inspection, micro-CT images and histological analysis. Data was analyzed by One-way ANOVA followed by Fisher’s Least significance difference test. A p-value less than 0.05 was considered to be significantly different between the groups. Results: Metformin with frHMGB1 significantly decreased HMGB1 levels and decreased the enhanced expression of IL-1β compared to frHMGB1 alone. Micro-CT images showed completely healed at day-90 post-surgery in frHMGB1 rats while Metformin inhibited frHMGB1 enhanced bone fracture healing as evidenced by the gap in the fractured bone area(Fig 2). Histology analysis indicated that frHMGB1 enhanced fractured bone healing as evidenced by high density of the cells was found in the bone fracture area (Fig. 3). Safranin O and fast green staining along with Masson’s Trichrome staining confirmed the frHMGB1 local injection promoted fractured bone healing (Fig. 4) as evidenced by high quality bone tissue formation with collagen type I (red staining) and collagen type III (blue staining) in the fractured bone wound area. Conclusion: Fully reduced HMGB1 treatment enhanced healing in a nonunion diabetic rat model. The fracture site had high density of cells, quality bone tissue, and enhanced production of collagen I and III. However, Metformin inhibited frHMGB1-induced healing enhancement. In diabetic rats, nonunion bone fracture was present with evidence of inflammation showing elevated levels of HMGB1 and IL-1β.FrHMGB1 treatment enhanced healing of nonunion bone fracture by promoting cell proliferation and migration, limited HMGB1 concentration, increased collagen production. Although Metformin injection slowed down the bone fracture healing by inhibiting frHMGB1activity, it also blocked HMGB1 release and reduced the inflammation caused by HMGB1

The superior regenerative potential of muscle-derived stem cells for articular cartilage repair is attributed to high cell survival and chondrogenic potential

Three populations of muscle-derived cells (PP1, PP3, and PP6) were isolated from mouse skeletal muscle using modified preplate technique and retrovirally transduced with BMP4/GFP.  In vitro, the PP1 cells (fibroblasts) proliferated significantly slower than the PP3 (myoblasts) and PP6 cells (muscle-derived stem cells); the PP1 and PP6 cells showed a superior rate of survival compared with PP3 cells under oxidative stress; and the PP6 cells showed significantly superior chondrogenic capabilities than PP1 and PP3 cells. In vivo, the PP6 cells promoted superior cartilage regeneration compared with the other muscle-derived cell populations. The cartilage defects in the PP6 group had significantly higher histological scores than those of the other muscle-derived cell groups, and GFP detection revealed that the transplanted PP6 cells showed superior in vivo cell survival and chondrogenic capabilities compared with the PP1 and PP3 cells. PP6 cells (muscle-derived stem cells) are superior to other primary muscle-derived cells for use as a cellular vehicle for BMP4-based ex vivo gene therapy to heal full-thickness osteo-chondral defects. The superiority of the PP6/muscle-derived stem cells appears to be attributable to a combination of increased rate of in vivo survival and superior chondrogenic differentiation capacity

Patient-Reported Outcome Measures After Surgical Management of Unstable Lisfranc Injuries in Athletes

Background: Athletes sustaining Lisfranc joint instability after a low-energy injury often undergo surgical fixation. Limited studies report validated patient-reported outcome measures (PROMs) for this specific patient population. Our purpose was to report PROMs of athletes experiencing instability after a low-energy Lisfranc injury and undergoing surgical fixation. Methods: Twenty-nine athletes (23 competitive, 6 recreational) sustained an unstable Lisfranc injury (14 acute, 15 chronic) and met our inclusion criteria. Injuries were classified as acute if surgically managed within 6 weeks. All athletes completed validated PROMs pre- and postoperatively. The cohort underwent various open reduction internal fixation methods. We evaluated outcomes with the Foot and Ankle Ability Measure (FAAM) activities of daily living (ADL) and sports subscales. Results: Fourteen of 29 (48%) athletes reported PROMs at ≥2 years with a median follow-up time of 44.5 months. Substantial improvement for both FAAM ADL (50% vs 93%; P  < .001) and sports (14.1% vs 80%; P  = .002) subscales were found, when comparing preoperative to postoperative scores at ≥2 years. Conclusion: This study provides outcomes information for the young athletic population that were treated operatively for low-energy Lisfranc injury with apparent joint instability. Based on the FAAM sports subscale, these patients on average improved between their 6-month evaluation and their final ≥2 years but still scored 80% of the possible 100%, which indicates continued but “slight” difficulty with lower extremity function. Level of Evidence: Level IV, case series

The Effects Of Protease-Activated Receptors 1 And 4 In Human Platelet Activation And Inflammation

Category: Basic Sciences/Biologics Introduction/Purpose: Tendon injuries occur frequently and cost billions of health care dollars annually. Recently, there has been an increase in the use of platelet-rich plasma (PRP) to treat tendon injuries. However, the efficacy of PRP treatment is controversial due to inconsistent results from human clinical trials. It is thought that variations in PRP preparation contribute to these inconsistencies. Specifically, platelets in PRP contain pro-angiogenic (e.g. VEGF) or anti-angiogenic (e.g. endostatin) factors, which may differentially affect the healing of tendon injuries. It is known that these factors are selectively released after platelet activation by specific receptors. Therefore, in this study we investigated the effect of protease-activated receptors 1 and 4 (PAR1 and PAR4) in platelet activation and inflammation. Methods: Platelet preparation – Human blood was obtained from 12 healthy donors and 9 ml of blood was mixed with 1 ml of 3.8% sodium citrate and centrifuged at 500g for 10 min. Then, the supernatant (PRP) without the buffy coat was centrifuged at 1000g for 10 min and the resulting pellet was washed in Tyrodes-HEPES buffer and centrifuged for 10 min at 1000g. Finally, platelets in the pellet was suspended in Tyrodes-HEPES buffer and used in experiments. Platelet activation – About 100 μl of platelet from above was activated with 5 μl 1 mM PAR1-activating peptide (PAR1-AP) or PAR4-activating peptide (PAR4-AP) at 25°C for 10 min. Then, the mixture was centrifuged at 1000g for 10 min, and the levels of VEGF, endostatin, IL-1RA and HMGB-1in the supernatant was determined by ELISA. Platelets without activators were used as controls. Results: PAR1 induced angiogenic effects in human platelets. PAR1 activated platelets released 3 times more VEGF than when activated with PAR4 (Fig. 1A). However, PAR4 activated platelets released 7 times more endostatin than the PAR1 activated platelets (Fig. 1B). Further, PAR1 induced anti-inflammatory effects in human platelets; it did not change IL-1R-A (Fig. 2A) but decreased HMGB-1 levels (Fig. 2B). In contrast, PAR4 stimulated inflammatory effects in human platelets by lowering IL-1-RA and increasing HMGB-1 levels. Conclusion: Our findings indicate that PAR1 induces angiogenetic and anti-inflammatory effects in human platelets, while PAR4 has anti-angiogenetic and inflammatory effects. Of significance is HMGB-1, which is constitutively expressed in the nuclei of most mammalian cells. Under cellular stress, HMGB1 is released into the extracellular matrix and activates the immune response thus acting as a danger-signal. Both PAR1 and PAR4 selectively regulated the release of VEGF and endostatin, and IL-1RA and HMGB-1 from human platelets. Therefore, the role of PAR1 and PAR4 on human platelet activation and inflammation should be considered prior to the use of PRP to treat tendon injuries

Kartogenin

Category: Basic Sciences/Biologics Introduction/Purpose: Tendon-bone junction (TBJ) injuries are very common and optimal treatment modalities are lacking. TBJ injuries often heal without formation of the fibrocartilage transition zone increasing the risk of rerupture. Current treatments only attach tendons to bones but do not promote healing or regeneration of the fibrocartilage zone. Therefore, we developed a novel approach to heal wounded TBJ using a small molecule called kartogenin (KGN). KGN was previously shown to induce chondrogenesis of tendon stem/progenitor cells (TSCs) in vitro and enhance wound healing in injured rat TBJs in vivo after short- term treatment (2 weeks). It is not known whether these effects can be maintained longterm. Therefore, we studied the long term (3 months) effects of KGN on achilles-tendon-bone junction healing. Methods: KGN stock was prepared in DMSO and diluted to 100 μM with PRP solution. PRP was obtained from the blood of Sprague–Dawley rats and the platelet concentration in PRP was adjusted to 3 times higher than that in whole blood. Thrombin (10 kU/ml) served as the PRP activator. 42 female rats (2.5 – 3 months old) were used. 1 mm diameter defects were created at the Achilles tendon-bone junction area in each hind leg of all rats. Rats were divided into 3 treatment groups: Group 1 (KGN+PRP): KGN (100 μM) +25 μl PRP + 5 μl thrombin; Group 2 (PRP): 25 μl PRP + 5 μl thrombin; and Group 3 (Control): 30 μl saline. All the rats were sacrificed at 3 months, and hind legs harvested for analyses. N= 6 hind legs were used for histological and qPCR analysis. N = 16 hind legs were used for mechanical testing. Results: Single treatment of KGN+PRP demonstrates improved TBJ healing (Fig. 1A) compared to PRP only and control groups (Fig. 1B, C). Fibrocartilage-like tissue regeneration occurred only in the KGN+PRP treated group (Fig. 2A). These results were further confirmed by qPCR, which showed significantly higher expression of cartilage-related genes (Data not shown). Furthermore, the cartilage-like transition zone in the KGN+PRP group also stained positive for Col-I, Col-II, Scx and Sox-9 indicating the regeneration of fibrocartilage tissues in the KGN+PRP treated TBJ (Fig. 3A, B). Mechanical testing showed that the KGN+PRP (47±12 N) and PRP (50±13 N) groups had significantly higher ultimate strength than the controls (39±16 N). There was no significant difference in ultimate strength between the KGN+PRP group and PRP group. Conclusion: In this study, we have shown that KGN injection enhanced healing of the wounded TBJ by promoting the formation of a fibrocartilage transition zone. Thus, KGN can be used as a mode of cell free therapy to promote regeneration of the fibrocartilage transition zone in injured TBJs. PRP alone enhanced healing of the TBJ but did not induce fibrocartilage regeneration. PRP + KGN are advantageous due to the numerous growth factors located within PRP and PRPs ability to serve as a KGN carrier. Future research is needed to optimize KGN and PRP dosage regimens and the optimal delivery methods

Kartogenin Enhances Tendon Graft And Bone Tunnel Healing In A Rat Model

Category: Basic Sciences/Biologics Introduction/Purpose: The normal tendon-bone junction (TBJ) is a strong structure protected by the fibrocartilage transition zone. This allows a gradual transition of mechanical forces between tendon and bone, thus decreasing stress-concentration effects. Healing of the TBJ interface after an injury is slow and even after healing the junction often lacks the transition zone. A study on human patients showed that even years after ACL reconstruction, patients had no fibrocartilage zone regeneration. This and other studies show that surgical repair alone does not restore the unique protective fibrocartilage transition zone. We have shown that KGN injection into injured rat Achilles tendon-bone junctions enhanced wound healing with restored fibrocartilage transition zone. Here, we examined the effects of KGN treatment, along with platelet-rich plasma (PRP), on tendon-bone tunnel healing in rats. Methods: KGN stock was prepared in DMSO and diluted to 100 μM with PRP. PRP was obtained from the blood of Sprague– Dawley rats and the platelet concentration in PRP was adjusted to 3 times over the baseline platelet concentration in whole blood. Thrombin (1 kU/mL), served as the PRP activator. 27 female rats (234˜268g) were used. A 1.5 mm tunnel was drilled at the distal end of tibia. Achilles tendon was resected and sutured in the tunnel. Rats were randomly divided into 1 of 3 treatment groups: Group A: 50µl KGN + PRP; Group B: 50µl PRP solution; Group C: control. Rats were sacrificed at 4, 8, and 12 weeks for histological analysis. Whole tibia with the tendon insertion were harvested, fixed in 10% formalin and decalcified in 10% EDTA. Tissue were sectioned and stained with Safranin O + Fast green, and immunostained for collagen types 1 and 2. Results: All animals were in good condition after surgery and complications were not present. Safranin O staining was higher in the KGN+PRP group than the other groups indicating more cartilage-like tissues regeneration in this group. Formation of the cartilage-like transitional zone was time dependent; i.e., it increased with increase in time (Fig. 1A-C). In contrast, both PRP and control groups had no cartilage-like tissues (Fig. 1D-I); in fact, some gaps in the control group were found in the tendon-bone interface after 4 and 8 weeks (Fig. 1G-I). Finally, the cartilage-like tissues in the KGN+PRP group also stained positive for both Col-1 and Col-2 indicating that these were fibrocartilage tissues (Fig. 2). Conclusion: Here we demonstrated that KGN promotes the formation of fibrocartilage-like interface between the tendon graft and bone tunnel. This result suggests that the delivery of KGN into the tendon-bone interface could be a promising, cell-free approach to augment the tendon-bone interface healing. PRP in this study, while not effective in promoting fibrocartilage formation of the interface in its own right, it functions as an effective carrier that supplies scaffolds and growth factors necessary for the enhancement of wound healing. Future research is required to determine the optimal KGN dosage regimens and the optimal delivery method (e.g. injection vs implantation)

Co-Morbidities Are Associated with Increased Cost, Infection Rates, and Duration of Treatment after Primary Achilles Tendon Repair

Category: Sports. Introduction/Purpose: The purpose of this study was to assess the rate of surgical site infection (SSI) and surgical irrigation and debridement (I&D) after primary Achilles tendon repair. Secondary objectives were to assess the potential effect(s) of medical comorbidities on cost and duration of treatment of SSI after Achilles tendon repair. Methods: De-identified patient insurance records within the government and private national insurance orthopaedic datasets were searched between 2005-2012. The Current Procedural Terminology (CPT) code was used to identify primary Achilles tendon repair and I&D. Subsequently, post-operative SSIs and comorbidities were examined by searching corresponding International Classification of Disease Ninth Revision, Clinical Modification (ICD-9-CM) codes. Results: 24,269 primary Achilles tendon repairs were identified. Overall, there was a significantly increased rate of SSI if a medical comorbidity was present at the time of surgery compared to those without a comorbidity (17.96% vs. 5.96%, p < 0.0001). Patients with diabetes and vascular complications had the highest SSI rate (OR 7.85, CI 6.25-9.86, p < 0.001), followed by peripheral vascular disease, diabetes with peripheral neuropathy, history of drug abuse, fluid and electrolyte abnormalities, obesity, and uncomplicated diabetes. There was higher rate of surgical I&D in patients with cardiac arrhythmias and uncomplicated hypertension. There was a significant increase in cost of SSI treatment ($6,004.09 vs.$4,184.62, p=0.006) and duration of treatment (8.41 days vs. 5.54 days, p < 0.001) if a medical comorbidity was present in Achilles tendon patients with SSI. Conclusion: An analysis of a large cohort of patients undergoing Achilles tendon reconstruction revealed that having certain medical comorbidities conferred a significantly greater risk for developing SSI, which increased both the cost of subsequent care and duration of treatment. Furthermore, with the advent of “value”/outcomes based care being linked to reimbursement, SSI is a one measure being used by the Centers for Medicare & Medicaid Services and private insures to determine appropriate orthopaedic care. Thus, patients with modifiable risk factors should be referred for medical management prior to surgery or deferred to a non-operative treatment program to reduce the risk of SSI after Achilles tendon repair

Adipose-Derived Mesenchymal Stem Cells Treated with Growth Differentiation Factor-5 Express Tendon-Specific Markers

A sensitive spectrophotometric method for the analysis of isoniazid, lisinopril dihydrate, amoxicillin trihydrate, ampicillin trihydrate, glucosamine sulfate, phenylpropanolamine hydrochloride and gabapentin is described. The analysis is based on the reaction of drug molecules with ninhydrin and sodium molybdate mixture to give Ruhemann's purple product with maximum absorbance (lambda(max)) at 570 nm. The statistical analysis of intra-day and inter-day estimation of drugs as well as comparison with reported methods demonstrated high precision and accuracy of the proposed method. The method was successfully applied to the analysis of pharmaceutical preparations. The procedure was suitable for quality control application