David M. Chowaniec

Platelet-Rich Plasma Differs According to Preparation Method and Human Variability

BACKGROUND Varying concentrations of blood components in platelet-rich plasma preparations may contribute to the variable results seen in recently published clinical studies. The purposes of this investigation were (1) to quantify the level of platelets, growth factors, red blood cells, and white blood cells in so-called one-step (clinically used commercial devices) and two-step separation systems and (2) to determine the influence of three separate blood draws on the resulting components of platelet-rich plasma. METHODS Three different platelet-rich plasma (PRP) separation methods (on blood samples from eight subjects with a mean age [and standard deviation] of 31.6 ± 10.9 years) were used: two single-spin processes (PRPLP and PRPHP) and a double-spin process (PRPDS) were evaluated for concentrations of platelets, red and white blood cells, and growth factors. Additionally, the effect of three repetitive blood draws on platelet-rich plasma components was evaluated. RESULTS The content and concentrations of platelets, white blood cells, and growth factors for each method of separation differed significantly. All separation techniques resulted in a significant increase in platelet concentration compared with native blood. Platelet and white blood-cell concentrations of the PRPHP procedure were significantly higher than platelet and white blood-cell concentrations produced by the so-called single-step PRPLP and the so-called two-step PRPDS procedures, although significant differences between PRPLP and PRPDS were not observed. Comparing the results of the three blood draws with regard to the reliability of platelet number and cell counts, wide variations of intra-individual numbers were observed. CONCLUSIONS Single-step procedures are capable of producing sufficient amounts of platelets for clinical usage. Within the evaluated procedures, platelet numbers and numbers of white blood cells differ significantly. The intra-individual results of platelet-rich plasma separations showed wide variations in platelet and cell numbers as well as levels of growth factors regardless of separation method.

The Positive Effects of Different Platelet-Rich Plasma Methods on Human Muscle, Bone, and Tendon Cells

Background: Clinical application of platelet-rich plasma (PRP) in the realm of orthopaedic sports medicine has yielded variable results. Differences in separation methods and variability of the individual may contribute to these variable results. Purpose: To compare the effects of different PRP separation methods on human bone, muscle, and tendon cells in an in vitro model. Study Design: Controlled laboratory study. Methods: Blood collected from 8 participants (mean ± SD age 31.6 ± 10.9 years) was used to obtain PRP preparations. Three different PRP separation methods were used: a single-spin process yielding a lower platelet concentration (PRPLP), a single-spin process yielding high platelet and white blood cell concentrations (PRPHP), and a double-spin that produces a higher platelet concentration and lower white blood cell concentration (PRPDS). Human bone, muscle, and tendon cells obtained from discarded tissue samples during shoulder surgery were placed into culture and treated with the 3 PRP preparations, control media (2% fetal bovine serum [FBS] and 10% FBS), and native blood. Radioactive thymidine assays were obtained to examine cell proliferation, and testing with enzyme-linked immunosorbent assay was used to determine growth factor concentrations. Results: Addition of PRPLP to osteocytes, myocytes, and tenocytes significantly increased cell proliferation (P ≤ .05) compared with the controls. Adding PRPDS to osteoblasts and tenocytes increased cell proliferation significantly (P ≤ .05), but no significance was shown for its addition to myocytes. The addition of PRPHP significantly increased cell proliferation compared with the controls only when added to tenocytes (P ≤ .05). Osteoblasts: Proliferation was significantly increased by addition of PRPLP compared with all controls (2% FBS, 10% FBS, native blood) (P ≤ .05). Addition of PRPDS led to significantly increased proliferation compared with all controls, native blood, and PRPHP (P ≤ .05). Proliferation was significantly less when PRPHP was added compared with PRPDS (P ≤ .05). Myocytes: Proliferation was significantly increased by addition of PRPLP compared with native blood (P ≤ .05). Adding PRPHP or PRPDS to myocytes showed no significant increase in proliferation compared with the controls or the other separations. Tenocytes: Proliferation was significantly increased by addition of PRPLP compared with all controls (2% FBS, 10% FBS, native blood) (P ≤ .05). Addition of PRPDS showed a significant increase compared with the controls and native blood. For tenocytes, there was a significant increase (P ≤ .05) seen when PRPHP was added compared with the controls and native blood but not compared with the other separations. Conclusion: The primary findings of this study suggest the application of different PRP separations may result in a potential beneficial effect on the clinically relevant target cells in vitro. However, it is unclear which platelet concentration or PRP preparation may be optimal for the treatment of various cell types. In addition, a “more is better” theory for the use of higher platelet concentrations cannot be supported. This study was not intended to prove efficacy but to provide a platform for future research to be built upon. Clinical Relevance: The utilization of different PRP separations may result in a potentially beneficial effect on the clinically relevant target cells in vitro, but it is unclear which platelet concentration or PRP preparation may be optimal for the treatment of various cell types.

Rapid Isolation of Human Stem Cells (Connective Tissue Progenitor Cells) From the Proximal Humerus During Arthroscopic Rotator Cuff Surgery

Background: Bone-to-tendon healing in the shoulder can be unpredictable. Biologic augmentation, through the implementation of adult mesenchymal stem cells, may improve this healing process. Purpose: The purpose of this study was to (1) arthroscopically obtain bone marrow aspirates from the proximal humerus during rotator cuff repair, (2) purify and concentrate the connective tissue progenitor cells (CTPs) in the operating room efficiently, and (3)confirm these are stem cells through their ability to differentiate into bone cells. We hypothesize that CTPs can be quickly and efficiently isolated from bone marrow during arthroscopic surgery and that these cells are capable of osteogenesis. Study Design: Cohort study; Level of evidence, 3; and Descriptive laboratory study. Methods: Bone marrow aspirates were harvested through the anchor tunnel of the humeral head during arthroscopic rotator cuff repair in 23 patients. Twenty-three matched controls were selected from a clinical registry to evaluate for increased incidence of complication. Connective tissue progenitor cells were isolated using 2 accepted methods and compared with a novel, rapid method designed for use in the operating room. Osteogenic potential was assessed by cytochemical and molecular analysis. Results: Reverse transcription polymerase chain reaction analysis and cellular staining confirmed the osteogenic potential of these CTPs. There was no statistical significant difference in the Single Assessment Numeric Evaluation score (aspirate, 86.3 ± 10.5; control, 83.6 ± 15.1; P = .54), range of motion measures (postoperative external rotation: aspirate, 65.0° ± 20.4°; control, 62.5° ± 17.1°; P = .67; postoperative forward elevation: aspirate, 163.0° ± 30.6°; control, 145.7° ± 41.4°; P = .12), or postoperative strength measures between groups (median, 5; range, 4-5 in the aspirate group compared with median, 5; range, 4-5 in the control group; P > .05). Conclusion: Connective tissue progenitor cells can be safely and efficiently aspirated from the proximal humerus using the anchor tunnel created during arthroscopic rotator cuff surgery. These cells may play an important role in cell-based therapies involving rotator cuff repair. Clinical Relevance: We have established a reliable, reproducible protocol for isolating CTPs in the operating room. These cells may have the potential to enhance the healing process after rotator cuff repair.

Corticosteroids and Local Anesthetics Decrease Positive Effects of Platelet-Rich Plasma: An In Vitro Study on Human Tendon Cells

PURPOSE To determine the effects of mixing anesthetics or corticosteroids with platelet-rich plasma (PRP) on human tenocytes in vitro. METHODS Two separate protocols (double spin and single spin) were used to obtain homologous PRP from the blood of 8 healthy volunteers. Discarded tendon acquired during biceps tenodesis served as tendon specimens for all experiments. After cell isolation, tenocytes were treated in culture with PRP alone or in combination with corticosteroids and/or anesthetics. Fetal bovine serum in concentrations of 2% and 10% served as controls. Cell exposure times of 5, 10, and 30 minutes were used. Radioactive thymidine and luminescence assays were obtained to examine cell proliferation and viability. RESULTS The presence of lidocaine, bupivacaine, or methylprednisolone resulted in significantly less proliferation than the negative 2% fetal bovine serum control (P < .05). When we compared groups, both lidocaine and bupivacaine had a greater inhibitory effect than methylprednisolone (P < .05). At all time points, viability was significantly decreased in the presence of lidocaine, bupivacaine, or methylprednisolone compared with the negative control (P < .05). CONCLUSIONS The addition of either anesthetics or corticosteroids to PRP resulted in statistically significant decreases in tenocyte proliferation and cell viability. These results suggest that incorporation of anesthetics or corticosteroids, either alone or in combination, with PRP injection may compromise the potentially beneficial in vitro effects of isolated PRP on tendon cells and compromise cell viability at the site of tendon injury. CLINICAL RELEVANCE Anesthetics or corticosteroids either alone or in combination should be used carefully to preserve the proposed positive effects of PRP in the treatment of tendon injury.

Biomechanical Comparison of Arthroscopic Repairs for Acromioclavicular Joint Instability Suture Button Systems Without Biological Augmentation

Background: Arthroscopic procedures for reconstruction of acromioclavicular (AC) joint separations are increasingly used in clinical practice. Multiple surgical techniques exist, but there are still few data on biomechanical performances of commonly used arthroscopic techniques and fixation methods. Hypothesis: Single and double clavicular tunnel reconstructions show comparable primary stability with a modified Weaver-Dunn procedure, and double tunnel constructs show superior horizontal stability. Study Design: Controlled laboratory study. Methods: The AC joints of 40 cadaveric shoulders were tested for anterior, posterior, and superior translation (70-N load) and maximal load to failure (superior) with the MTS 858 Bionix II Servohydraulic testing system. Shoulders were assigned to 4 groups: (1) native (n = 18), (2) coracoclavicular (CC) reconstruction with 1 clavicular and 1 coracoid tunnel (SCT) fixed with a suture pulley and 2 buttons (n = 8), (3) CC reconstruction with 2 clavicular and 1 coracoid tunnel (DCT) fixed with a suture pulley and 3 buttons (n = 8), and (4) modified Weaver-Dunn reconstruction (n = 6). Results: Native specimens showed a mean anterior translation of 7.92 mm (±1.69 mm), a mean posterior translation of 7.84 mm (±2.09 mm), and a superior translation of 4.28 mm (±1.81 mm). Maximal load to failure was 579.44 N (±148.01 N). The SCT technique showed a mean anterior translation of 5.81 mm (±1.16 mm), posterior translation of 8.30 mm (±1.94 mm), and a superior translation of 2.28 mm (±0.52 mm). The maximal load to failure was 591.35 N (±231.17 N). Anterior and superior translations were significantly less compared with the native specimen (P = .005 and P = .003). The DCT technique had an anterior translation of 4.68 mm (±0.6 mm), posterior translation of 6.85 mm (±0.83 mm), and superior translation of 2.09 mm (±0.86 mm). The mean maximal load to failure was 651.16 N (±226.93 N). Anterior and superior translations were significantly less compared with the native specimens (P = .000 and P = .001). No statistically significant differences were shown between SCT and DCT reconstruction for all measurements (P > .05). One reconstruction of the modified Weaver-Dunn procedure failed directly after mounting it into the testing device. The remaining 5 showed a mean anterior translation of 11.36 mm (±3.17 mm), a mean posterior translation of 13.51 mm (±2.21 mm), and a mean superior translation of 3.31 mm (±0.47 mm). Anterior and posterior translations were significantly increased compared with the native specimen (P = .019 and P = .000). The mean maximal load to failure measured 311.13 N (±52.2 N) and was significantly less compared with the native specimen (P = .000). The Weaver-Dunn technique showed significantly less maximal load to failure and more anterior and posterior translation compared with SCT and DCT (P ≤ .05). Conclusion: Isolated reconstruction of the CC ligaments using single and double clavicular tunnel techniques results in a high load to failure for superior translation, which is equal to the native stability, and less translation in all 3 directions as well as higher superior stability when compared with the modified Weaver-Dunn procedure. A potential drawback is the risk of coracoid fracture, as the high load to failure of the device may exceed load to failure of cortical bone prior to device breakage. Clinical Relevance: Single clavicular tunnel arthroscopic reconstructions of the coracoacromial ligaments show good biomechanical results.

Material Properties and Composition of Soft-Tissue Fixation

Surgical interference screws and suture anchors for attaching soft tissue, such as ligaments and tendons, to bone are routinely used in arthroscopic surgery and sports medicine. Interference screw fixation provides a press fit between bone, graft/tendon, and screw and is frequently used to attach replacement ligaments in tunnels drilled for anterior and posterior cruciate ligament reconstruction. Suture anchors are used in surgical procedures wherein it is necessary for a surgeon to attach (tie) tissue to the surface of the bone, for example, during joint reconstruction and ligament repair or replacement. The composition of these implants ranges from metals to polymers and composites. Typically, because of the relatively large amount of torque that must be applied during insertion, these screws are constructed from metal. However, interference screws and suture anchors have also been constructed from bioabsorbable polymers and composites. The ideal material would (1) provide adequate mechanical fixation, (2) completely degrade once no longer needed, and (3) be completely replaced by bone. Because no material has been shown to be superior for all applications, the surgeon must weigh the advantages and disadvantages of each to evaluate the optimum material for a given application and patient. The purpose of this article is to present a comprehensive review of the commercially available interference screws and suture anchors, with an emphasis on implant composition, interaction, and design. This article provides the orthopaedic surgeon with a background on biomaterials, specifically those used in interference screws and suture anchors. Because there is no material that is perfect for all surgical situations, this review can be used to make educated decisions on a case-by-case basis.

Sequential Resection of the Distal Clavicle and Its Effects on Horizontal Acromioclavicular Joint Translation

Background: Reconstructions of the coracoclavicular (CC) ligament in an open or arthroscopically assisted procedure are often combined with a resection of the distal clavicle to prevent or treat osteoarthritic degenerations of the acromioclavicular (AC) joint. However, increased horizontal translation leading to symptomatic instability may be associated with resection of the distal clavicle. Hypothesis: Horizontal translation increases in direct correlation to subsequent resection of the distal clavicle. Study Design: Controlled laboratory study. Methods: Twelve fresh-frozen specimens (mean age, 82.0 ± 5 years) were examined using a servohydraulic testing system. A 70-N force was applied in the anterior, posterior, and superior directions, and the translations were quantified for all directions. After native testing, sequential resection of the distal clavicle at 5 and 10 mm was performed after incision of the inferior capsule, and anterior-posterior loads were retested. Finally, the AC ligaments were incised, and repeated testing was performed. Results: Native mean (± standard deviation) anterior translation was 7.90 ± 2.62 mm. After section of the inferior AC capsule, anterior translation was 8.05 ± 2.62 mm (P = 1.0). After clavicle resection of 5 mm, anterior translation was 10.75 ± 2.38 mm (P = .122), and after resection of 10 mm, anterior translation was significantly increased to 11.6 ± 2.37 mm (P = .012). Complete AC capsule sectioning led to significantly greater amounts of anterior translation (12.12 ± 3.0 mm; P = .003). Posterior translation for the native specimen was 8.88 ± 2.63 mm. Sectioning of the inferior AC capsule resulted in 9.21 ± 2.71-mm translation (P = 1.0). After clavicle resection of 5 mm, posterior translation was 10.42 ± 2.42 mm (P = 1.0), and after 10-mm resection, it was 11.31 ± 2.86 mm (P = .39). Sectioning of the complete AC capsule led to significantly greater amounts of posterior translation (12.31 ± 3.12 mm; P = .043). Conclusion: Ten millimeters of resection increased anterior translation of the distal clavicle with both the superior and posterior AC capsules as well as the CC ligaments intact. Both anterior and posterior translations increased after 10-mm resection and complete AC capsule sectioning. Clinical Relevance: These results suggest that resection of the distal clavicle leads to increased horizontal translation despite an intact superior and posterior AC capsule. Only sparing resection of the distal clavicle should be performed and only if strictly indicated. Violation of the AC capsule further increases horizontal translation and should therefore be avoided, or if indicated in AC joint dislocations, a reconstruction of the AC capsule should be considered.

Biomechanical evaluation of classic solid and novel all-soft suture anchors for glenoid labral repair.

PURPOSE To evaluate the biomechanical performance of an all-soft suture anchor (JuggerKnot; Biomet, Warsaw, IN) in comparison with a classic solid suture anchor (2.4-mm biocomposite SutureTak; Arthrex, Naples, FL) in an in vitro labral repair model. METHODS We dissected 12 cadaveric shoulders (mean age, 61 ± 9.4 years), leaving the labrum intact, and bone mineral density was obtained (mean, 0.375 ± 0.06 g/cm(3)). Simulated labral tears were made at the anteroinferior and posteroinferior edges of the labrum. Repairs used 2 all-soft suture anchors (JuggerKnot) or 2 solid anchors with free, high-strength No. 2 suture (FiberWire; Arthrex) spanning the operative construct to load the repair. Differential variable reluctance transducers were used to measure labral displacement for each specimen. The testing protocol consisted of a preconditioning phase at 10 N for 10 cycles (1 Hz) and then a final load-to-failure testing at a rate of 3 mm/min. Labral displacement of 2 mm was determined as the primary outcome. RESULTS There was no statistical difference (P = .22) in ultimate load to failure and displacement at ultimate failure (anchor pullout) between the all-soft JuggerKnot (146.0 ± 43.0 N and 19.8 ± 5.4 mm, respectively) and the solid SutureTak (171.9 ± 52.6 N and 22.3 ± 6.8 mm, respectively). The solid anchor had a significantly higher ultimate load at 2 mm of labral displacement than the all-soft suture anchor (84.1 ± 19.0 N and 39.2 ± 10.6 N, respectively; P < .001). CONCLUSIONS Whereas both the solid SutureTak and the all-soft JuggerKnot displayed similar results on ultimate load-to-failure testing, the solid anchor required significantly greater load for 2 mm of labral displacement than the all-soft anchor. CLINICAL RELEVANCE The all-soft anchor (JuggerKnot) is similar in biomechanical performance to the classic solid anchor (SutureTak) with the exception of load at 2 mm of labral displacement, suggesting micromotion of the device.

In vitro changes in human tenocyte cultures obtained from proximal biceps tendon: multiple passages result in changes in routine cell markers

PurposeResults of in vitro cell models are commonly used to promote new therapies (e.g., platelet-rich plasma), and clinicians have to be aware of the specific limitations of such models. To gain a sufficient and effective cell load, many current in vitro models use cells multiplied through various passages. This is especially important in tendon-like cell (TLC) models, since native tendon tissue is not available unlimited and contains limited amount of tenocytes. The purpose was to determine the occurrence of phenotypic changes following extended monolayer culture of TLCs, according to cell-passage number.MethodsTendon samples were obtained from 15 healthy patients undergoing biceps tenodesis. Tendons were digested and cultured (monolayer) for six passages. Tendon-specific markers (collagens I and III, decorin, tenascin-C, and tenomodulin) and their histology were analyzed using gene expression and protein content assays. Native cells, the cells cultured and cells passaged one to six times were analyzed at each passage.ResultsGene expression of types I and III collagen of cultured TLCs significantly decreased after two passages. Gene expression of decorin, tenascin-C, and tenomodulin exhibited a trend of decreased gene expression with increased passage. Protein levels of types I and III collagen and decorin decreased after four passages.ConclusionsThe significant findings let conclude that tenocyte-like cells obtained from human LHB can be maintained in monolayer culture at low passages, before the signs of phenotypic drift are present. But researchers must be aware of rapid phenotypic drift at higher passage numbers. Therefore, only cells within the first 3 passages should be used as a precaution for in vitro monolayer cell models, and one has to be aware of the phenotypic changes if TLCs passaged multiple times are used. The clinical relevance of this data is that understanding of in vitro TLC models, and their limitations may finally help the clinician to judge the potential of experimental data of new biologic treatment options.

Biomechanical Evaluation of Effect of Coracoid Tunnel Placement on Load to Failure of Fixation During Repair of Acromioclavicular Joint Dislocations

PURPOSE To evaluate the effect of entry and exit points of the coracoid tunnel on load to failure and mode of failure, to reduce the incidence of coracoid fractures and acromioclavicular joint repair failures. METHODS This study investigates 5 tunnel placements based on different entry and exit points in the coracoid process: center-center orientation represents perfect placement of the bone tunnel and served as perfect tunnel placement in our study. Four common errors in drilling were then tested and acted as the experimental groups in our study (medial-center, center-medial, lateral-center, and center-lateral). Using 35 cadaveric shoulders (mean age, 68.0 ± 13.0 years), we tested these 5 tunnel orientations using a single repair technique (cortical button) loaded to failure on an MTS 858 Servohydraulic test system (MTS Systems, Eden Prairie, MN). A control group of 7 cadaveric shoulders without the presence of a coracoid tunnel was also tested to determine the type of fracture pattern that occurred. RESULTS The coracoids without tunnel drilling fractured in patterns similar to traumatic coracoid injuries. With regard to the 5 tunnel groups, it was found that the loads to failure with center-center and medial-center tunnel placement were significantly higher than those with center-medial, center-lateral, and lateral-center tunnel placement. The failure modes of the former were primarily within the repair constructs, whereas those of the latter were primarily due to bony failure. CONCLUSIONS Our biomechanical results showed a higher peak load to failure with a center-center or medial-center tunnel orientation, which may lessen the risk of coracoid fracture during drilling with a 6-mm cannulated drill bit. CLINICAL RELEVANCE Proper trajectory of the drill during formation of a coracoid bone tunnel can help reduce the risk of coracoid process fracture and repair failure.