Fibrin Clots Maintain the Viability and Proliferative Capacity of Human Mesenchymal Stem Cells: An In Vitro Study.

BACKGROUND: Augmentation of soft-tissue repairs with an autologous fibrin clot has been used clinically for nearly four decades; however, fibrin clots tend to produce an abundance of scar tissue, which is known to inhibit soft-tissue regeneration. Mesenchymal stem cells (MSCs) embedded in fibrin clots before repair could reduce scar tissue deposition and facilitate soft-tissue regeneration. To our knowledge, no published studies have directly evaluated the viability or bioactivity of MSCs in fresh human fibrin clots over time. The purpose of this study was to evaluate the viability and bioactivity of human MSCs inside human fibrin clots over time in nutritive and non-nutritive culture media. QUESTIONS/PURPOSES: We hypothesized that human MSCs would (1) be captured inside fibrin clots and retain their proliferative capacity, (2) remain viable for at least 7 days in the fibrin clots, (3) maintain their proliferative capacity for at least 7 days in the fibrin clots without evidence of active apoptosis, and (4) display similar viability and proliferative capacity when cultured in a non-nutritive medium over the same time periods. METHODS: Twelve patients (mean age 33.7 years; range 4-72 years) who underwent elective knee surgery were approached between February 2016 and October 2017; all patients agreed to participate and were enrolled. MSCs isolated from human skeletal muscle and banked after prior studies were used for this analysis. On the day of surgery and after expansion of the MSC population, 3-mL aliquots of phosphate-buffered saline containing approximately 600,000 labeled with anti-green fluorescent protein (GFP) antibodies were transported to the operating room, mixed in 30 mL of venous blood from each enrolled patient, and stirred at 95 rpm for 10 minutes to create MSC-embedded fibrin clots. The fibrin clots were transported to the laboratory with their residual blood for analysis. Eleven samples were analyzed after exclusion of one sample because of a processing error. MSC capture was qualitatively demonstrated by enzymatically digesting half of each clot specimen, thus releasing GFP-positive MSCs into culture. The released MSCs were allowed to culture for 7 days. Manual counting of GFP-positive MSCs was performed at 2, 3, 4, and 7 days using an inverted microscope at 100 x magnification to document the change in the number of GFP-positive MSCs over time. The intact remaining half of each clot specimen was immediately placed in proliferation media and allowed to culture for 7 days. On Days 1, 2, 3, 4, and 7, a small portion of the clot was excised, flash-frozen, cryosectioned (8-µm thickness), and immunostained with antibodies specific to GFP, Ki67 (indicative of active proliferation), and cleaved caspase-3 ([CC3]; indicative of active apoptosis). Using an inverted microscope, we obtained MSC cell counts manually at time zero and after 1, 2, 3, 4, and 7 days of culture. Intact fresh clot specimens were immediately divided in half; one half was placed in nutritive (proliferation media) and the other was placed in non-nutritive (saline) media for 1, 2, 3, 4, and 7 days. At each timepoint, specimens were processed in an identical manner as described above, and a portion of each clot specimen was excised, immediately flash-frozen with liquid nitrogen, cryosectioned (8-µm thickness), and visualized at 200 x using an inverted microscope. The numbers of stain-positive MSCs per field of view, per culture condition, per timepoint, and per antibody stain type were counted manually for a quantitative analysis. Raw data were statistically compared using t-tests, and time-based correlations were assessed using Pearson's correlation coefficients. Two-tailed p values of less than 0.05 (assuming unequal variance) were considered statistically significant. RESULTS: Green fluorescence, indicative of viable GFP-positive MSCs, was absent in all residual blood samples after 48 hours of culturing; GFP-positive MSCs were visualized after enzymatic digestion of clot matrices. The number of GFP-positive MSCs per field of view increased between the 2-day and 7-day timepoints (mean 5.4 ± 1.5; 95% confidence interval, 4.7-6.1 versus mean 17.0 ± 13.6; 95% CI, 10.4-23.5, respectively; p = 0.029). Viable GFP-positive MSCs were present in each clot cryosection at each timepoint up to 7 days of culturing (mean 6.2 ± 4.3; 95% CI, 5.8-6.6). There were no differences in MSC counts between any of the timepoints. There was no visible evidence of GFP +/CC3 + double-positive MSCs. Combining all timepoints, there were 0.34 ± 0.70 (95% CI, 0.25-0.43) GFP+/Ki67+ double-positive MSCs per field of view. The mitotic indices at time zero and Day 7 were 7.5% ± 13.4% (95% CI, 3.0%-12.0%) and 7.2% ± 14.3% (95% CI, 3.3%-12,1%), respectively (p = 0.923). There was no visible evidence of GFP +/CC3 + double-positive MSCs (active apoptosis) at any timepoint. For active proliferation in saline-cultured fibrin clots, we found averages of 0.1 ± 0.3 (95% CI, 0.0-0.2) and 0.4 ± 0.9 (95% CI, 0.0-0.8) GFP/Ki67 double-positive MSCs at time zero and Day 7, respectively (p = 0.499). The mitotic indices in saline culture at time zero and Day 7 were 2.9% ± 8.4% (95% CI, 0.0%-5.8%) and 9.1% ± 20.7% (95% CI, 1.2%-17.0%; p = 0.144). There was no visible evidence of GFP +/CC3 + double-positive MSCs (active apoptosis) at any timepoint in either culturing condition. CONCLUSION: These preliminary in vitro results show that human MSCs mixed in unclotted fresh human venous blood were nearly completely captured in fibrin clots and that seeded MSCs were capable of maintaining their viability, proliferation capacity, and osteogenic differentiation capacity in the fibrin clot for up to 7 days, independent of external sources of nutrition. CLINICAL RELEVANCE: Fresh human fibrin clots have been used clinically for more than 30 years to improve soft-tissue healing, albeit with scar tissue. Our results demonstrate that allogenic human MSCs, which reduce soft-tissue scarring, can be captured and remain active inside human fibrin clots, even in the absence a nutritive culture medium.

Editorial Commentary: Posterolateral Corner Injuries in Elite Athletes Remain a Challenging Diagnostic Problem.

Posterolateral corner injuries are rare, and the examination to quantitate these injuries is very challenging. Careful examination comparing the involved and uninvolved knees, considering stress radiographs, and combining this with magnetic resonance imaging findings is currently our most accurate way to grade these injuries.

Editorial Commentary: "There, It Fits!"-Justifying Nonsignificant P Values.

The increasing emphasis placed on value-based medicine has become a powerful motivating factor that has driven the performance and publication of more comparative clinical outcome studies. Although these endeavors are well intentioned and significant progress has been made in our field over the past few decades, I believe that we need both to re-emphasize the importance of results derived from meaningful study designs and to avoid the notion that nonsignificant P values represent study failure.

Variability Between Full-Length Lateral Radiographs and Standard Short Knee Radiographs When Evaluating Posterior Tibial Slope in Revision ACL Patients.

Background: Increased posterior tibial slope (PTS) has been identified as a risk factor for failure after anterior cruciate ligament (ACL) reconstruction. Correction of PTS may improve outcomes after revision ACL reconstruction. There are conflicting reports demonstrating the measurement of the PTS on standard short knee (SSK) radiographs versus full-length lateral (FLL) radiographs including the entire tibia. Purpose/Hypothesis: To compare PTS measurements between SSK and FLL radiographs in patients who failed primary ACL reconstruction. It was hypothesized that there would be high variability between the SSK and FLL radiographic measurements. Study Design: Cohort study (diagnosis); Level of evidence, 2. Methods: The medial and lateral PTS were measured on the SSK and FLL radiographs of 33 patients with failed primary ACL reconstructions. All measurements were performed by 2 trained independent observers (A.A.M., J.S.), and inter- and intraobserver reliability were calculated using the intraclass correlation coefficient (ICC). Measurements recorded by the observer with the higher intraobserver ICC were used for comparison of the PTS on SSK versus FLL radiographs. Results: Both the inter- and the intraobserver reliability values of the PTS measurements were excellent. There was a significant difference in mean PTS on the medial plateau as measured on the SSK and FLL radiographs (11.2°± 5.3° vs 12.5°± 4.6°; P = .03), with the FLL radiographs demonstrating higher PTS. There was also a significant difference in the mean PTS on the lateral plateau as measured on SSK versus FLL radiographs (10.7°± 4.3° vs 12.2°± 4°, respectively; P = .01), with the FLL radiographs demonstrating higher PTS. Notably, 66.67% of the absolute measurements for PTS on the medial plateau differed by ≥2°, with variability as high as 8.5°. Conclusion: Results indicated that FLL and SSK radiographs are not interchangeable measurements for PTS associated with failed ACL reconstruction. Because FLL radiographs demonstrate less variability than SSK radiographs, we recommend obtaining them to evaluate these complex patients.

Single-Strand "Short Isometric Construct" Medial Collateral Ligament Reconstruction Restores Valgus and Rotational Stability While Isolated Deep MCL and Superficial MCL Reconstruction Do Not.

BACKGROUND: Historical MCL (medial collateral ligament) reconstruction (MCLR) techniques have focused on the superficial MCL (sMCL) to restore valgus stability while frequently ignoring the importance of the deep MCL (dMCL) in controlling tibial external rotation. The recent recognition of the medial ligament complex importance has multiple studies revisiting medial anatomy and questioning contemporary MCLR techniques. PURPOSE: To assess whether (1) an isolated sMCL reconstruction (sMCLR), (2) an isolated dMCL reconstruction (dMCLR), or (3) a novel single-strand short isometric construct (SIC) would restore translational and rotational stability to a knee with a dMCL and sMCL injury. STUDY DESIGN: Controlled laboratory study. METHODS: Biomechanical testing was performed on 14 fresh-frozen cadaveric knee specimens using a custom multiaxial knee activity simulator. The specimens were divided into 2 groups. The first group was tested in 4 states: intact, after sectioning the sMCL and dMCL, isolated sMCLR, and isolated dMCLR. The second group was tested in 3 states: intact, after sectioning the sMCL and dMCL, and after single-strand SIC reconstruction (SICR). In each state, 4 loading conditions were applied at 0°, 20°, 40°, 60°, and 90° of knee flexion: 8-N·m valgus torque, 5-N·m external rotation torque, 90-N anterior drawer, and combined 90-N anterior drawer plus 5-N·m tibial external rotation torque. Anterior translation, valgus rotation, and external rotation of the knee were measured for each state and loading condition using an optical motion capture system. RESULTS: sMCL and dMCL transection resulted in increased laxity for all loading conditions at all flexion angles. Isolated dMCLR restored external rotation stability to intact levels throughout all degrees of flexion, yet valgus stability was restored only at 0° of flexion. Isolated sMCLR restored valgus and external rotation stability at 0°, 20°, and 40° of flexion but not at 60° or 90° of flexion. Single-strand SICR restored valgus and external rotation stability at all flexion angles. In the combined anterior drawer plus external rotation test, isolated dMCL and single-strand SICR restored stability to the intact level at all flexion angles, while the isolated sMCL restored stability at 20° and 40° of flexion but not at 60° or 90° of flexion. CONCLUSION: In the cadaveric model, single-strand SICR restored valgus and rotational stability throughout the range of motion. dMCLR restored rotational stability to the knee throughout the range of motion but did not restore valgus stability. Isolated sMCLR restored external rotation and valgus stability in early flexion. CLINICAL RELEVANCE: In patients with anteromedial rotatory instability in the knee, neither an sMCLR nor a dMCLR is sufficient to restore stability.

Relationship Between Bone Bruise Volume and Patient Outcomes After ACL Reconstruction.

Background: Subchondral bone injuries, or bone bruises, are commonly observed on magnetic resonance imaging (MRI) after anterior cruciate ligament (ACL) injury. The current relationship between bone bruise volume and postsurgical outcomes remains poorly understood. Purpose: To examine the influence of bone bruise volume on self-reported and objective functional outcomes at the time of return to play and 2 years following ACL reconstruction. Study Design: Cohort study; Level of evidence, 3. Methods: Clinical, surgical, and demographic data were obtained for a sample of convenience utilizing a single-surgeon ACL database (n = 1396). For 60 participants, femoral and tibial bone bruise volumes were estimated from preoperative MRI. Data obtained at the time of return to play included International Knee Documentation Committee (IKDC-2000) score, ACL-Return to Sport after Injury (ACL-RSI) score, and performance on an objective functional performance battery. Two-year follow-up data included graft reinjury rate, level of return to sport/activity, and self-reported knee function using the Single Assessment Numeric Evaluation (SANE). The forward stepwise linear regression was used to determine the relationship between bone bruise volume and patient function. Results: The distribution of bone bruise injuries was as follows: lateral femoral condyle (76.7%), lateral tibial plateau (88.3%), medial femoral condyle (21.7%), and medial tibial plateau (26.7%). Mean total bone bruise volume of all compartments was 7065.7 ± 6226.6 mm3. At the 2-year follow up, there were no significant associations between total bone bruise volume and time of return to play (P = .832), IKDC-2000 score (P = .200), ACL-RSI score (P = .370), or SANE score (P = .179). Conclusion: The lateral tibial plateau was the most frequent site to sustain bone bruise injury. Preoperative bone bruise volume was not associated with delayed time to return to sport or self-reported outcomes at time of return to play or at 2 years postoperatively. Registration: NCT03704376 (ClinicalTrials.gov identifier).

Comparison of 2 femoral tunnel locations in anatomic single-bundle anterior cruciate ligament reconstruction: a biomechanical study.

PURPOSE: To evaluate knee stability after anterior cruciate ligament (ACL) reconstruction using 2 modern clinically relevant single-bundle constructs. METHODS: Two arthroscopic ACL reconstructions were performed on 6 fresh-frozen human cadaveric knees using bone-patellar tendon-bone autografts. The tibial tunnel was centered in the anatomic tibial footprint. The femoral tunnel was reamed through the anteromedial (AM) portal and centered alternately in either the AM portion of the femoral footprint (center-AM) or the center of the femoral footprint (center-center). Two external loading conditions were applied: (1) a 134-N anterior tibial load and (2) a 10-Nm valgus load combined with a 5-Nm internal tibial torque. Resulting kinematics were determined under 4 conditions: (1) ACL intact, (2) ACL deficient, (3) center-AM reconstruction, and (4) center-center reconstruction. RESULTS: In response to anterior tibial loading, anterior translation was similar in the ACL-intact knee and the 2 reconstructions at 0° to 60° of flexion but was greater in the reconstructed specimens at 90°. In response to the complex rotatory load, internal tibial rotation (ITR) at 30° of flexion was slightly greater in center-AM knees compared with ACL-intact knees (11.0° ± 0.6° v 10.5° ± 0.6°, P = .03). At other angles tested, ITR in both reconstructions was similar to the ACL-intact knee (P > .05). When we compared the 2 reconstruction alternatives, however, center-center knees exhibited greater resistance to ITR at all angles (P < .05). CONCLUSION: Anatomic single-bundle ACL reconstruction performed with the femoral tunnel placed through the AM portal restores translational and rotational knee stability to an extent that closely approximates the ACL-intact condition. When compared with the AM femoral tunnel position, a femoral tunnel positioned in the anatomic center of the femoral origin of the ACL may further improve rotatory stability without sacrificing anterior stability. CLINICAL RELEVANCE: This study provides additional biomechanical evidence in support of anatomic single-bundle ACL reconstruction with tunnels positioned in the center of the femoral and tibial footprints.

Utilizing Hip Abduction Strength to Body-Weight Ratios in Return to Sport Decision-Making After ACL Reconstruction.

BACKGROUND: Despite the association between hip abduction weakness and non-contact anterior cruciate ligament (ACL) injury, hip abduction strength is rarely considered in return to sport decision-making following ACL reconstruction (ACLR). HYPOTHESIS/PURPOSE: The purpose of this study was to compare self-reported function, objective functional test performance, and re-injury rates in patients with high (≥33%) versus low (<33% ) isometric hip abduction strength to body weight (BW) ratios when returning to activity following ACLR. STUDY DESIGN: Cohort study. METHODS: Data were gathered from a single-surgeon database and included baseline demographics. Clinical outcomes were assessed at the time of release to activity and included self-reported outcomes and a functional testing battery. Isometric hip abduction strength was obtained using a handheld dynamometer. Groups were dichotomized into those with low vs high strength to BW ratios. Two-year follow-up was performed using the single assessment numeric evaluation (SANE). Data were analyzed using univariate general linear models with an alpha level of .05. RESULTS: Of the 528 enrolled patients, 364 (68.9%) demonstrated a low strength to BW ratio. Baseline comparisons revealed more females and higher BMI (P <.05) in the <33% group. At release to activity, the <33% BW group demonstrated lower International Knee Documentation Committee survey scores (88.2 ± 13.6 vs 93.5 ± 10.3, P<.01), ACL-Return to Sport After Injury (76.2 ± 15.4 vs 88.5 ± 16.9, P<.01) scores, and isokinetic hamstring peak torque (P=.04). At 2-years, the <33% group reported lower SANE scores (83.3 ± 21.1 vs 92.83 ± 11.4, P=.05) with no significant differences in re-injuries. CONCLUSION: Patients with low hip abduction strength to BW ratios demonstrated lower subjective function, psychological readiness, and isokinetic hamstring peak torque when completing functional testing following ACLR. Subjective deficits remained at 2-years. LEVEL OF EVIDENCE: Level 3. KEY TERMS: ACL injury, hip abduction strength, return to sport, strength ratio. CLINICAL RELEVANCE: Assessing isometric hip abduction strength to body weight ratio may be beneficial in determining readiness to return to sport following ACL reconstruction. WHAT IS KNOWN ABOUT THE SUBJECT: Three prospective studies have provided conflicting evidence regarding the relationship between hip abduction strength and ACL injury. A clinical cut-point of hip abduction strength:BW ratio <35.4% has been suggested to identify athletes at risk of sustaining a non-contact ACL injury. To our knowledge no studies have examined isometric hip abduction strength:BW ratios in athletes attempting to return to sport following ACLR. WHAT THIS STUDY ADDS TO EXISTING KNOWLEDGE: This study examines the potential for hip abduction strength:BW ratio to be included as an additional metric in return to sport testing batteries.

Platelet-Rich Plasma Augmentation of Meniscal Repair in the Setting of Anterior Cruciate Ligament Reconstruction.

BACKGROUND: The increasing incidence of anterior cruciate ligament (ACL) and meniscal injuries has led to strong interest in discovering new methods to enhance the biological healing response of these tissues. Platelet-rich plasma (PRP) contains various growth factors associated with a positive healing response, but few existing clinical studies are available to determine the risks and benefits of these therapies. PURPOSE: To determine the effects of intraoperative PRP on postoperative knee function and complications at 2 years after ACL reconstruction with meniscal repair. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A retrospective matched case-control study was conducted between 2013 and 2017 using a single surgeon database of 1014 patients undergoing primary ACL reconstruction with concomitant meniscal repair, resulting in 324 patients (162 PRP patients and 162 control patients) who met the study criteria. Patients were matched by age, sex, graft type, and meniscal injury. The Single Assessment Numeric Evaluation (SANE) was administered at 2 years, and injury surveillance was conducted. Secondary outcomes included the time to return to activity (months), self-reported knee function (International Knee Documentation Committee [IKDC] score), functional performance testing (knee range of motion, single-leg balance, single-leg hopping, agility testing), and postoperative complications (graft failure, infection, loss of motion [requiring repeat arthroscopy for lysis of adhesions], venous thrombosis, etc). Univariate models were used for between-group comparisons, and alpha was set at .05 for all analyses. RESULTS: No differences were found in SANE knee function scores between the PRP and matched-control groups at 2 years (91.6 ± 11.2 vs 92.4 ± 10.6, respectively; P = .599). Additionally, no differences were reported between groups for self-reported function (IKDC score, 87.6 ± 13.3 vs 88.1 ± 12.6; P = .952), functional performance testing (P > .05), and timing of return to activity (7.8 ± 1.9 vs 8.0 ± 1.9 months; P = .765). The PRP group demonstrated a higher rate of postoperative knee motion loss compared with the control group (13.6% vs 4.6%; P < .001). No other differences were observed in postoperative complications (P > .05). CONCLUSION: The added use of intraoperative PRP did not improve self-reported knee function, functional performance, and timing of return to activity for patients undergoing ACL reconstruction with meniscal repair. Furthermore, the use of PRP may have negative consequences for regaining knee range of motion after surgery. On the basis of these data, surgeons should cautiously consider the application of PRP when planning surgery for intra-articular injuries of the knee. REGISTRATION: NCT03704376 (ClinicalTrials.gov identifier).

The Influence of Mode-of-Injury on Psychological Readiness for Return-To-Sport Following Anterior Cruciate Ligament Reconstruction: A Matched-Controlled Study.

BACKGROUND: Self-efficacy and fear of re-injury have been documented as factors related to an athlete's ability to return-to-sport after anterior cruciate ligament (ACL) reconstruction. The purpose of this study was to compare psychological readiness between athletes injured in their primary mode of sport versus those injured outside of their primary sport following ACL reconstruction. HYPOTHESIS: Athletes sustaining 'in-sport' injuries will demonstrate poorer psychological readiness when compared their matched counterparts injured outside of their primary sport. STUDY DESIGN: Case-Control Study. METHODS: A single-surgeon database of 638 patients following ACL reconstruction was used to conduct a matched case-control analysis. Psychological readiness was examined 16-weeks postoperatively using the ACL-Return to Sport after Injury (ACL-RSI) questionnaire with subgroup analyses for the 'emotional', 'confidence' and 'injury-risk' subscales. Subject matching was performed for baseline patient and surgical demographics. All statistical comparisons were performed using a one-way (group) analysis variance (ANOVA) at a significance level of α = .05. RESULTS: Ninety-two matched patients (49 'in-sport' injuries, 43 'out-of-sport' injuries) were included in the final analysis. The 'in-sport' group exhibited significantly lower total ACL-RSI scores (55.3 ±12.9 versus 60.8 ±11.6, t = 2.747, P < .001) when compared to the 'out-of-sport' group. Subscale comparisons indicated lower 'emotional' (P < .016) and higher 'injury risk' (P < .001) psychological constructs for 'in-sport' athletes versus 'out-of-sport' athletes. No differences were found between groups for the 'confidence' subscale (P = .987). CONCLUSIONS: Athletes sustaining 'in-sport' ACL injuries demonstrated poorer psychological readiness when compared to athletes injured outside their primary sport when in preparation for return-to-sport activities following ACL reconstruction. CLINICAL RELEVANCE: Clinicians should consider the potential impact of mode of injury on psychological readiness when returning athletes to sport after ACL reconstruction.

Multiple Ligament Knee Injuries: Does the Knee Dislocation Classification Predict the Type of Surgical Management?

This study aims to evaluate relationships among multiple ligament knee injury (MLKI) patterns as classified according to the knee dislocation (KD) classification and the types of surgical management pursued. We hypothesized that the KD classification would not be predictive of the types of surgical management, and that categorizing injuries according to additional injury features such as structure, chronicity, grade, and topographic location would be predictive of the types of surgical management. This is a Retrospective cohort study. This study was conducted at a level I trauma center with a 150-mile coverage radius. Query of our billing database was performed using combinations of 43 billing codes (International Classification of Diseases [ICD] 9, ICD-10, and Current Procedural Terminology) to identify patients from 2011 to 2015 who underwent operative management for MLKIs. There were operative or nonoperative treatment for individual ligamentous injuries, repair, or reconstruction of individual ligamentous injuries, and staging or nonstaging or nonstaging of each surgical procedure. The main outcome was the nature and timing of clinical management for specific ligamentous injury patterns. In total, 287 patients were included in this study; there were 199 males (69.3%), the mean age was 30.2 years (SD: 14.0), and the mean BMI was 28.8 kg/m2 (SD: 7.4). There were 212 injuries (73.9%) categorized as either KD-I or KD-V. The KD classification alone was not predictive of surgery timing, staging, or any type of intervention for any injured ligament (p > 0.05). Recategorization of injury patterns according to structure, chronicity, grade, and location revealed the following: partial non-ACL injuries were more frequently repaired primarily (p < 0.001), distal medial-sided injuries were more frequently treated operatively than proximal medial-sided injuries (odds ratio [OR] = 24.7; p <0.0001), and staging was more frequent for combined PCL-lateral injuries (OR = 1.3; p = 0.003) and nonavulsive fractures (OR = 1.2; p = 0.0009). The KD classification in isolation was not predictive of any surgical management strategy. Surgical management was predictable when specifying the grade and topographic location of each ligamentous injury. This is a Level IV, retrospective cohort study.

The Beneficial Effect of an Intra-articular Injection of Losartan on Microfracture-Mediated Cartilage Repair Is Dose Dependent.

BACKGROUND: A previous publication demonstrated that the oral intake of losartan promoted microfracture-mediated hyaline-like cartilage repair in osteochondral defects of a rabbit knee model. However, an intra-articular (IA) injection of losartan may have direct beneficial effects on cartilage repair and has not been studied. PURPOSE: To determine the dosage and beneficial effects of an IA injection of losartan on microfracture-mediated cartilage repair and normal cartilage homeostasis. STUDY DESIGN: Controlled laboratory study. METHODS: Rabbits were divided into 5 groups (n = 6 each): a microfracture group (MFX group) and 4 different losartan treatment groups that received varying doses of IA losartan (0.1, 1, 10, and 100 mg per knee). An osteochondral defect (5 mm) was created in the trochlear groove cartilage of 1 limb in each rabbit, and 5 microfracture perforations were made in the osteochondral defect. Both the injured and the contralateral knee joints were injected with IA losartan immediately after microfracture and at 2 and 4 weeks after surgery. Rabbits were sacrificed at 6 weeks after surgery for analysis including gross observation, micro-computed tomography, histology, and reverse transcription quantitative polymerase chain reaction. RESULTS: Micro-computed tomography and gross observation demonstrated comparable subchondral bone healing and hyaline-like cartilage morphology in the 0.1-, 1-, and 10-mg losartan groups relative to the MFX group. Conversely, the 100-mg losartan group showed neither bony defect healing nor cartilage repair. Histology revealed higher O'Driscoll scores and hyaline-like cartilage regeneration in the 1-mg losartan group compared with the MFX group. In contrast, the 100-mg losartan group showed the lowest histology score and no cartilage repair. An IA injection of losartan at the doses of 0.1, 1, and 10 mg did not cause adverse effects on uninjured cartilage, while the 100-mg dose induced cartilage damage. Quantitative polymerase chain reaction results showed downregulation of the transforming growth factor ß (TGF-ß) signaling pathway after IA losartan injection. CONCLUSION: An IA injection of losartan at the dose of 1 mg was most effective for the enhancement of microfracture-mediated cartilage repair without adversely affecting uninjured cartilage. Conversely, a high dose (100 mg) IA injection of losartan inhibited cartilage repair in the osteochondral defect and was chondrotoxic to normal articular cartilage. CLINICAL RELEVANCE: An IA injection of losartan at an optimal dosage represents a novel microfracture enhancement therapy and warrants a clinical trial for future clinical applications.

Intra-articular Injection of Bevacizumab Enhances Bone Marrow Stimulation-Mediated Cartilage Repair in a Rabbit Osteochondral Defect Model.

BACKGROUND: Bone marrow stimulation (BMS) via microfracture historically has been a first-line treatment for articular cartilage lesions. However, BMS has become less favorable because of resulting fibrocartilage formation. Previous studies have shown that angiogenesis blockade promotes cartilage repair. Bevacizumab is a Food and Drug Administration-approved medication used clinically to prevent angiogenesis. HYPOTHESIS: The intra-articular injection of bevacizumab would prevent angiogenesis after BMS and lead to improved cartilage repair with more hyaline-like cartilage. STUDY DESIGN: Controlled laboratory study. METHODS: The dose of bevacizumab was first optimized in a rabbit osteochondral defect model with BMS. Then, 48 rabbits (n = 8/group/time point) were divided into 3 groups: osteochondral defect (defect), osteochondral defect + BMS (BMS group), and osteochondral defect + BMS + bevacizumab intra-articular injection (bevacizumab group). Rabbits were sacrificed at either 6 or 12 weeks after surgery. Three-dimensional (3D) micro-computed tomography (microCT), macroscope score, modified O'Driscoll histology scores, collagen type 2, Herovici staining, and hematoxylin and eosin staining were performed. Angiogenesis markers were also evaluated. RESULTS: The intra-articular dose of 12.5 mg/0.5 mL bevacizumab was found to be effective without deleteriously affecting the subchondral bone. Intra-articular injection of bevacizumab resulted in significantly improved cartilage repair for the bevacizumab group compared with the BMS or the defect group based on 3D microCT, the macroscope score (both P < .05), the modified O'Driscoll histology score (P = .0034 and P = .019 vs defect and BMS groups, respectively), collagen type 2, Herovici staining, and hematoxylin and eosin staining at 6 weeks. Cartilage in the bevacizumab group had significantly more hyaline cartilage than did that in other groups. At 12 weeks, the cartilage layer regenerated in all groups; however, the bevacizumab group showed more hyaline-like morphology, as demonstrated by microCT, histology scores (P < .001 and .0225 vs defect and BMS groups, respectively), histology, and immunohistochemistry. The bevacizumab injection did not significantly change mRNA expressions of smooth muscle actin, vascular endothelial growth factor, or hypoxia-inducible factor-1 alpha. CONCLUSION: Intra-articular injection of bevacizumab significantly enhanced the quality and quantity of hyaline-like cartilage after BMS in a rabbit model. Future large-animal and human studies are necessary to evaluate the clinical effect of this therapy, which may lead to improved BMS outcomes and thus the durability of the regenerated cartilage. CLINICAL RELEVANCE: The use of bevacizumab may be an important clinical adjunct to improve BMS-mediated cartilage repair.

Anatomic double-bundle anterior cruciate ligament reconstruction: kinematics and knee flexion angle-graft tension relation.

PURPOSE: The purpose of this study was to compare the bundle tension curves and resultant knee kinematics between 2 tensioning protocols in anatomic double-bundle anterior cruciate ligament (ACL) reconstruction. METHODS: Anatomic double-bundle ACL reconstruction was performed in 7 male cadaveric knees. Each graft was tensioned to 22 N under 2 conditions: (1) both bundles tensioned at 20 degrees of knee flexion (20/20 protocol) or (2) posterolateral (PL) bundle tensioned at 15 degrees and anteromedial (AM) bundle at 45 degrees (45/15 protocol). Knee kinematics were recorded in response to anterior and combined rotatory loads in the intact, ACL-deficient, and reconstructed states. Bundle tension was recorded dynamically with knee motion and during each loading test. RESULTS: Tensioning both bundles at 20 degrees of knee flexion resulted in a reciprocal bundle tension pattern that was not statistically different; the PL bundle tension was greater than the AM bundle tension in full extension, and the AM bundle tension was greater than the PL bundle tension from 25 degrees to 120 degrees. In the second tensioning protocol, the AM bundle tension was significantly greater than the PL bundle tension at all flexion angles. Both tensioning protocols restored normal knee kinematics. CONCLUSIONS: Bundle-tensioning protocol is a variable that has a significant effect on the bundle-loading patterns in double-bundle ACL reconstruction. The 20/20 protocol resulted in AM and PL bundle-loading patterns that were equivalent during dynamic testing, whereas the 45/15 protocol led to excessive tension in the AM bundle in full extension. We recommend equal tensioning of both bundles with the knee at 20 degrees of flexion to restore relatively normal tension curves in each bundle and to avoid excessive stress on the AM bundle. CLINICAL RELEVANCE: In double-bundle ACL reconstruction, there is no consensus regarding bundle-tensioning protocols. This study provides data on the individual bundle tension curves that result from 2 commonly used tensioning protocols. These data will assist clinicians as the technique and application of double-bundle ACL reconstruction move forward.

Quantitative Assessment of In Vivo Human Anterior Cruciate Ligament Autograft Remodeling: A 3-Dimensional UTE-T2* Imaging Study.

BACKGROUND: The timing of return to play after anterior cruciate ligament (ACL) reconstruction is still controversial due to uncertainty of true ACL graft state at the time of RTP. Recent work utilizing ultra-short echo T2* (UTE-T2*) magnetic resonance imaging (MRI) as a scanner-independent method to objectively and non-invasively assess the status of in vivo ACL graft remodeling has produced promising results. PURPOSE/HYPOTHESIS: The purpose of this study was to prospectively and noninvasively investigate longitudinal changes in T2* within ACL autografts at incremental time points up to 12 months after primary ACL reconstruction in human patients. We hypothesized that (1) T2* would increase from baseline and initially exceed that of the intact contralateral ACL, followed by a gradual decline as the graft undergoes remodeling, and (2) remodeling would occur in a region-dependent manner. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Twelve patients (age range, 14-45 years) who underwent primary ACL reconstruction with semitendinosus tendon or bone-patellar tendon-bone autograft (with or without meniscal repair) were enrolled. Patients with a history of previous injury or surgery to either knee were excluded. Patients returned for UTE MRI at 1, 3, 6, 9, and 12 months after ACL reconstruction. Imaging at 1 month included the contralateral knee. MRI pulse sequences included high-resolution 3-dimensional gradient echo sequence and a 4-echo T2-UTE sequence (slice thickness, 1 mm; repetition time, 20 ms; echo time, 0.3, 3.3, 6.3, and 9.3 ms). All slices containing the intra-articular ACL were segmented from high-resolution sequences to generate volumetric regions of interest (ROIs). ROIs were divided into proximal/distal and core/peripheral sub-ROIs using standardized methods, followed by voxel-to-voxel registration to generate T2* maps at each time point. This process was repeated by a second reviewer for interobserver reliability. Statistical differences in mean T2* values and mean ratios of T2*inj/T2*intact (ie, injured knee to intact knee) among the ROIs and sub-ROIs were assessed using repeated measures and one-way analyses of variance. P < .05 represented statistical significance. RESULTS: Twelve patients enrolled in this prospective study, 2 withdrew, and ultimately 10 patients were included in the analysis (n = 7, semitendinosus tendon; n = 3, bone-patellar tendon-bone). Interobserver reliability for T2* values was good to excellent (intraclass correlation coefficient, 0.84; 95% CI, 0.59-0.94; P < .001). T2* values increased from 5.5 ± 2.1 ms (mean ± SD) at 1 month to 10.0 ± 2.9 ms at 6 months (P = .001), followed by a decline to 8.1 ± 2.0 ms at 12 months (P = .129, vs 1 month; P = .094, vs 6 months). Similarly, mean T2*inj/T2*intact ratios increased from 62.8% ± 22.9% at 1 month to 111.1% ± 23.9% at 6 months (P = .001), followed by a decline to 92.8% ± 29.8% at 12 months (P = .110, vs 1 month; P = .086, vs 6 months). Sub-ROIs exhibited similar increases in T2* until reaching a peak at 6 months, followed by a gradual decline until the 12-month time point. There were no statistically significant differences among the sub-ROIs (P > .05). CONCLUSION: In this preliminary study, T2* values for ACL autografts exhibited a statistically significant increase of 82% between 1 and 6 months, followed by an approximate 19% decline in T2* values between 6 and 12 months. In the future, UTE-T2* MRI may provide unique insights into the condition of remodeling ACL grafts and may improve our ability to noninvasively assess graft maturity before return to play.

Biologically Regulated Marrow Stimulation by Blocking TGF-ß1 With Losartan Oral Administration Results in Hyaline-like Cartilage Repair: A Rabbit Osteochondral Defect Model.

BACKGROUND: Microfracture or bone marrow stimulation (BMS) is often the first choice for clinical treatment of cartilage injuries; however, fibrocartilage, not pure hyaline cartilage, has been reported because of the development of fibrosis in the repair tissue. Transforming growth factor ß1 (TGF-ß1), which can promote fibrosis, can be inhibited by losartan and potentially be used to reduce fibrocartilage. HYPOTHESIS: Blocking TGF-ß1 would improve cartilage healing in a rabbit knee BMS model via decreasing the amount of fibrocartilage and increasing hyaline-like cartilage formation. STUDY DESIGN: Controlled laboratory study. METHODS: An osteochondral defect was made in the patellar groove of 48 New Zealand White rabbits. The rabbits were divided into 3 groups: a defect group (defect only), a BMS group (osteochondral defect + BMS), and a BMS + losartan group (osteochondral defect + BMS + losartan). For the rabbits in the BMS + losartan group, losartan was administrated orally from the day after surgery through the day of euthanasia. Rabbits were sacrificed 6 or 12 weeks postoperatively. Macroscopic appearance, microcomputed tomography, histological assessment, and TGF-ß1 signaling pathway were evaluated at 6 and 12 weeks postoperatively. RESULTS: The macroscopic assessment of the repair revealed that the BMS + losartan group was superior to the other groups tested. Microcomputed tomography showed superior healing of the bony defect in the BMS + losartan group in comparison with the other groups. Histologically, fibrosis in the repair tissue of the BMS + losartan group was significantly reduced when compared with the other groups. Results obtained with the modified O'Driscoll International Cartilage Repair Society grading system yielded significantly superior scores in the BMS + losartan group as compared with both the defect group and the BMS group (F value: 15.8, P < .001, P = .012, respectively). TGF-ß1 signaling and TGF-ß-activated kinase 1 of the BMS + losartan group were significantly suppressed in the synovial tissues. CONCLUSION: By blocking TGF-ß1 with losartan, the repair cartilage tissue after BMS was superior to the other groups and consisted primarily of hyaline cartilage. These results should be easily translated to the clinic because losartan is a Food and Drug Administration-approved drug and it can be combined with the BMS technique for optimal repair of chondral defects. CLINICAL RELEVANCE: Biologically regulated marrow stimulation by blocking TGF-ß1 (oral intake of losartan) provides superior repair via decreasing fibrocartilage formation and resulting in hyaline-like cartilage as compared with outcomes from BMS only.

Achilles allograft reconstruction of a chronic complete proximal hamstring rupture.

Complete rupture of the origin of the hamstrings is an uncommon injury. Primary surgical repair is the treatment of choice, but in some not possible. We present a case of an avid cyclist who had significant disability from a 6-year-old injury. He underwent reconstruction with Achilles allograft and suture anchors. With the knee flexed to 90 degrees and after extensive mobilization, the retracted musculotendinous unit would not reach the ischial tuberosity. Two suture anchors were placed in the ischial tuberosity after soft tissue debridement. One limb of suture from each anchor was placed in the end of an Achilles allograft with a locking suture, with the other end used to pull the end of the graft to the ischial tuberosity. An additional suture was placed in running fashion down each side of the graft. Distally, the graft was fanned out and attached to the retracted hamstrings with interrupted sutures with the knee at 40 degrees and maximal proximal pull on the hamstrings. The patient outcome was excellent. He resumed high level cycling by 6 months after surgery with no symptoms. Isokinetic testing demonstrated a hamstring deficit of 25% at 60 degrees/s, and 20% at 180 degrees/s at 8 months.

Subsequent Surgery for Loss of Motion After Anterior Cruciate Ligament Reconstruction Does Not Influence Function at 2 Years: A Matched Case-Control Analysis.

BACKGROUND: Loss of motion (LOM) remains a common complication after anterior cruciate ligament (ACL) reconstruction and can be detrimental to patient outcomes after surgery. LOM is multifactorial, but nonsurgical and surgical solutions to this complex problem are available. A paucity of quality data exists evaluating clinical outcomes after the surgical treatment of patients with LOM after ACL reconstruction. HYPOTHESIS: Patients undergoing surgical lysis of adhesions and manipulation under anesthesia for LOM after ACL reconstruction will exhibit decreased function, lower outcome scores, and delayed time of release to play when compared with matched controls without LOM. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A database of 1572 patients undergoing ACL reconstruction was sampled from 2013 to 2017 to identify a total of 58 patients (LOM group [n = 29] vs matched control group [n = 29]). Group comparisons were examined for patients requiring a second surgical procedure for LOM versus matched controls after ACL reconstruction for differences in surgical timing, self-reported International Knee Disability Committee scores, objective function at release to play, and subjective knee function at 2 years with the Single Assessment Numeric Evaluation. The risk of a type I error was set at α = .05 for all statistical analyses. RESULTS: Patients who underwent lysis of adhesions and manipulation under anesthesia for LOM after ACL reconstruction exhibited no differences in Single Assessment Numeric Evaluation knee function at 2 years when compared with matched controls (85.8 ± 14.9 vs 88.0 ± 10.8, P = .606). All patients met release-to-play criteria. Only International Knee Disability Committee scores (P = .046) and single-legged hop testing (P = .050) reached statistically significant differences, with higher scores in the control group. There was no difference in the time to release to play (P = .034) or level of participation (P = .180) between the control and surgical groups. Subjective function scores at 2 years were not significantly different between groups. Tourniquet time during the index ACL reconstruction was shorter in the control group (P = .034). CONCLUSION: The findings of this study suggest that patients who undergo surgical treatment for LOM after ACL reconstruction can release to play at similar times but display relative deficits in single-legged-hop symmetry and lower self-reported function when compared with matched controls. Longer surgical times may increase the risk for LOM after ACL reconstruction. REGISTRATION: NCT03704376 (ClinicalTrials.gov identifier).

Potential Usefulness of Losartan as an Antifibrotic Agent and Adjunct to Platelet-Rich Plasma Therapy to Improve Muscle Healing and Cartilage Repair and Prevent Adhesion Formation.

Postoperative tissue fibrosis represents a major complication in orthopedics. Transforming growth factor beta 1 is a key molecule in the development of postoperative fibrosis. High concentrations of transforming growth factor beta 1 have also been implicated in various diseases. Agents that counteract the actions of transforming growth factor beta 1 have been investigated as potential antifibrotic medications and as adjunct treatment to platelet-rich plasma injections (increased amounts of transforming growth factor beta 1) to improve their effectiveness and/or safety profile. Losartan blocks transforming growth factor beta 1 action and has attracted special interest in orthopedic research that focuses on how to reduce the risk of postoperative fibrosis. [Orthopedics. 2018; 41(5):e591-e597.].

Influences of donor and host age on human muscle-derived stem cell-mediated bone regeneration.

BACKGROUND: Human muscle-derived stem cells (hMDSCs) have been shown to regenerate bone efficiently when they were transduced with Lenti-viral bone morphogenetic protein 2 (LBMP2). However, whether the age of hMDSCs and the animal host affect the bone regeneration capacity of hMDSCs and mechanism are unknown which prompted the current study. METHODS: We isolated three gender-matched young and old populations of skeletal muscle stem cells, and tested the influence of cells' age on in vitro osteogenic differentiation using pellet culture before and after Lenti-BMP2/green fluorescent protein (GFP) transduction. We further investigated effects of the age of hMDSCs and animal host on hMDSC-mediated bone regeneration in a critical-size calvarial bone defect model in vivo. Micro-computer tomography (CT), histology, and immunohistochemistry were used to evaluate osteogenic differentiation and mineralization in vitro and bone regeneration in vivo. Western blot, quantitative polymerase chain reaction (PCR), and oxidative stress assay were performed to detect the effects of age of hMDSCs on cell survival and osteogenic-related genes. Serum insulin-like growth factor 1 (IGF1) and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured with an enzyme-linked immunosorbent assay (ELISA). RESULTS: We found LBMP2/GFP transduction significantly enhanced osteogenic differentiation of hMDSCs in vitro, regardless of donor age. We also found old were as efficient as young LBMP2/GFP-transduced hMDSCs for regenerating functional bone in young and old mice. These findings correlated with lower phosphorylated p38MAPK expression and similar expression levels of cell survival genes and osteogenic-related genes in old hMDSCs relative to young hMDSCs. Old cells exhibited equivalent resistance to oxidative stress. However, both young and old donor cells regenerated less bone in old than young hosts. Impaired bone regeneration in older hosts was associated with high bone remodeling due to higher serum levels of RANKL and lower level of IGF-1. CONCLUSION: hMDSC-mediated bone regeneration was not impaired by donor age when hMDSCs were transduced with LBMP2/GFP, but the age of the host adversely affected hMDSC-mediated bone regeneration. Regardless of donor and host age, hMDSCs formed functional bone, suggesting a promising cell resource for bone regeneration.