==== Front PLoS One PLoS One plos PLOS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0287922 PONE-D-22-35213 Research Article Biology and Life Sciences Organisms Eukaryota Animals Vertebrates Amniotes Mammals Dogs Biology and Life Sciences Zoology Animals Vertebrates Amniotes Mammals Dogs Medicine and Health Sciences Rheumatology Arthritis Osteoarthritis Medicine and Health Sciences Surgical and Invasive Medical Procedures Biology and Life Sciences Bioengineering Biotechnology Medical Devices and Equipment Medical Implants Engineering and Technology Bioengineering Biotechnology Medical Devices and Equipment Medical Implants Medicine and Health Sciences Medical Devices and Equipment Medical Implants Medicine and Health Sciences Pharmacology Drugs Antimicrobials Antibiotics Biology and Life Sciences Microbiology Microbial Control Antimicrobials Antibiotics Biology and Life Sciences Cell Biology Cellular Types Animal Cells Blood Cells White Blood Cells Biology and Life Sciences Cell Biology Cellular Types Animal Cells Immune Cells White Blood Cells Biology and Life Sciences Immunology Immune Cells White Blood Cells Medicine and Health Sciences Immunology Immune Cells White Blood Cells Medicine and Health Sciences Health Care Health Care Providers Physicians Surgeons People and Places Population Groupings Professions Medical Personnel Physicians Surgeons Engineering and Technology Industrial Engineering Process Engineering Industrial Processes Manufacturing Processes Surface Treatments Multifactorial assessment of leukocyte reduced platelet rich plasma injection in dogs undergoing tibial plateau leveling osteotomy: A retrospective study Assessment of PRP injection in dogs undergoing tibial plateau leveling osteotomy https://orcid.org/0000-0002-0962-8537 Aryazand Yazdan Data curation Formal analysis Investigation Project administration Writing – original draft Writing – review & editing 1 ¤ https://orcid.org/0000-0003-4623-3582 Buote Nicole J. Conceptualization Formal analysis Methodology Resources Writing – review & editing 2 * Hsieh YuHung Data curation Formal analysis Validation Writing – original draft 1 Hayashi Kei Formal analysis Funding acquisition Methodology Writing – original draft 2 Rosselli Desiree Formal analysis Supervision Writing – original draft 1 1 VCA West Los Angeles, Los Angeles, California, United States of America 2 Department of Clinical Sciences, Small Animal Surgery Section, Cornell University College of Veterinary Medicine, Ithaca, New York, United States of America Abdel-Wanis Mohamed El-Sayed Editor Sohag University Faculty of Medicine, EGYPT Competing Interests: The authors have declared that no competing interests exist. ¤ Current address: VCA Veterinary Specialists of the Valley, Woodland Hills, California, United States of America * E-mail: njb235@cornell.edu 30 6 2023 2023 18 6 e028792228 12 2022 15 6 2023 © 2023 Aryazand et al 2023 Aryazand et al https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This study assessed the effects of concurrent intra-articular injection and Tibial Plateau Leveling Osteotomy (TPLO) plate surface treatment with leukoreduced platelet rich plasma (lPRP) on outcomes of dogs undergoing TPLO. A retrospective study of medical records for cases presenting from January 2018 to December 2020 was performed. Client-owned dogs with naturally occurring cranial cruciate ligament rupture that underwent TPLO surgery were divided into two groups. The lPRP group included cases that underwent intra-articular injection and plate surface treatment at the time of their TPLO. The control group (C) underwent TPLO without PRP treatment. Data analyzed included: presence of surgical site infection, implant removal rate, degree of change in OA progression score, lameness score progression and radiographic bone healing. The short- and long-term complication rate, hospitalization and antibiotic therapy were also compared between the groups. Descriptive statistics, comparison analyses (Chi square test, t-test, Fisher’s exact test) and multi-level logistic regression models were used for statistical analysis. A total of 110 cases met the study inclusion criteria: 54 = lPRP, 56 = C. There were no significant differences between groups with regard to gender, age, presence of meniscal tear, weight, or body condition score. Significant findings included: improved radiographic healing of the osteotomy in the lPRP group, improved global OA scores in the lPRP group, and improved lameness score at recheck examination in the lPRP group. There was no significant difference between the lPRP and C group with regard to surgical site infection and implant removal rate. Concurrent intra-articular injection and plate surface treatment with leukocyte reduced PRP at the time of TPLO, is beneficial in slowing the progression of OA, hastening the radiographic evidence of osteotomy healing, and improved lameness score on recheck examination. Leukocyte reduced PRP was not a significant factor in reducing SSI or implant removal rate. The authors received no specific funding for this work. Data AvailabilityThe code and data for this study is posted to https://github.com/stats-matt/PRP-study. Data Availability The code and data for this study is posted to https://github.com/stats-matt/PRP-study. ==== Body pmcIntroduction Cranial cruciate ligament (CrCL) rupture is the most common injury to the canine stifle and the leading cause of lameness in dogs [1]. Although there are variety of treatment options available to address CrCL rupture in dogs, Tibial Plateau Leveling Osteotomy (TPLO) remains the most commonly performed surgical procedure in the United States [2]. While TPLO generally has excellent predictable outcomes [3], there is evidence that osteoarthritis (OA) still progresses over time [4, 5]. Stabilization of the CrCl deficient stifle has been reported to slow, but not halt the progression of OA [5–7]. Infection is one of the most common complications following TPLO. The surgical site infection rate following TPLO ranges 3–15% [8–11]. Increased infection rate following TPLO may be due to a number of factors such as: surgeon’s experience, breed, duration of anesthesia, and performing bilateral simultaneous TPLO [11]. Reported TPLO implant removal rate due to infection is 3.5–7.5% [11, 12]. The sequelae of surgical site infection or implant removal surgery include increased owner dissatisfaction, financial cost, and patient morbidity [2, 12]. A variety of strategies have been proposed to decrease TPLO post-operative infection rate, such as the use post-operative antibiotics, the use of strict shaving and prepping protocols, and adhesive drapes (IO-ban surgical adhesive 3M, Canada) [13]. The use of postoperative antibiotic therapy has been debated as a factor that could decrease TPLO infection rate [9, 10] though a recently published literature review, found little evidence to support this strategy [14]. Platelet rich plasma (PRP) has been demonstrated to have antibacterial activity including against methicillin-resistant infected skin wounds in dogs [15, 16]. PRP is created after centrifugation of autologous blood and contains a platelet concentration of at least 3–5 times that of the patient’s peripheral blood [17]. Intra-articular PRP injections have also shown favorable results in reducing inflammation following CrCL rupture in dogs [18]. PRP has been shown to possess anti-inflammatory properties [19] and works as a stimulator of bone healing [20, 21]. Interestingly, intra-articular injections of PRP have been effective in slowing the progression of osteoarthritis (OA) in both humans [22, 23] and dogs [24], particularly in the canine stifle [18, 24]. The commercially available veterinary PRP products can be classified based on leukocyte count. Leukocyte increased products may elicit an inflammatory reaction following administration because leukocytes produce primarily pro-inflammatory cytokines [19, 25]. Leukocyte reduced (leukoreduced) PRP products, on the other hand, contain negligible number of leukocytes and are therefore less likely to cause a pro-inflammatory reaction [18, 19, 26]. Leukoreduced PRP injections modulate inflammation in joints by stimulating anabolism in the tissues, reducing catabolism, and enhancing viscoelastic properties [25–27]. Clinically, multiple injections of leukoreduced PRP in a CrCL deficient stifle, have been proven to improve pain and lameness scores in dogs [28]. Veterinary literature on the effects of PRP injection following CrCL rupture is currently limited. One study evaluated proximal tibial osteotomy healing when PRP was injected into the osteotomy at the time of surgery and found no significant difference in osteotomy healing time [29]. Another study investigated the effects of multiple intra-articular PRP injections on pain scores and functional outcome in an experimental CrCL deficient model in dogs. This study demonstrated improved scores for both variables with PRP injections [28]. To the author’s knowledge, there is no study assessing either surgical site infection rates or OA progression in stifles receiving intraoperative leukoreduced PRP (lPRP) injection at the time of TPLO in dogs. The objectives of our study were to assess the effects of concurrent intra-articular injection and TPLO plate surface treatment with leukocyte reduced platelet rich plasma (lPRP), at the time of TPLO, on surgical site infection rate, implant removal rate, OA progression score, lameness score and radiographic bone healing. We hypothesized that dogs treated with lPRP at the time of TPLO would: have lower incidence of surgical site infection, lower implant removal rate, decreased OA progression score, improved lameness score and equivalent osteotomy healing compared to controls. Material and methods Study design: Case-control retrospective case series Dogs undergoing TPLO for CrCL insufficiency from January 2018 to December 2020 were retrospectively included in our data set. Medical records were reviewed, and cases were divided into two groups based on surgeon preference and owner wishes. The lPRP group (lPRP) consisted of patients receiving intra-articular injection of lPRP (autologous conditioned plasma by Arthrex, Naples, Florida) as well as TPLO plate surface treatment. The control group (C) patients underwent TPLO without PRP injection or plate surface treatment. This study received VCA administrative approval but did not require full IACUC approval as it was retrospective in nature and previously collected data may be used in retrospective studies without IACUC approval. Inclusion/Exclusion criteria Inclusion criteria was any dog undergoing TPLO regardless of meniscus status within the mentioned time frame that had not previously received any PRP products. A minimum of 6 months follow-up was required for inclusion. Any long-term complication, incision infection, implant removal, persistent lameness, and whether a contralateral TPLO was performed during the follow up period was documented. Staged bilateral TPLOs were included in both groups. Exclusion criteria consisted of bilateral simultaneous TPLO procedures, dogs undergoing any other orthopedic procedure concurrently, dogs diagnosed with any non-CrCL related orthopedic or neurologic condition affecting gait, incomplete medical records, and dogs without examination follow up (either at our hospital or the primary veterinary office) for at least 6 months. Data collection Surgical and patient data retrieved from the medical records included: sex, age, date of TPLO procedure, duration of hospitalization, body weight in kilograms, body condition score (BCS, scale ranging from 1 = emaciated, 5 = ideal, to 9 = morbidly obese) [30], affected limb (right or left), visual lameness score prior to surgery (ranging from grade 0 = no lameness noted, grade 1 = intermittent, mild weight bearing lameness, grade 2 = obvious, moderate, weight bearing lameness, grade 3 = toe touching lameness, grade 4 = Non-weight bearing lameness) [31], pre-surgical tibial plateau angle (TPA), Pre-surgical platelet count of lPRP g, post-surgical TPA, subjective intra-operative synovitis assessment (mild, moderate, severe), intra-operative OA assessment (mid, moderate, severe), TPLO plate size and type (3.5mm broad, 3.5mm standard, 3.5mm mini, 2.7mm), volume of PRP injected (if noted), administration of local injection of bupivacaine liposome injectable suspension (NOCITA, Elanco Animal Health, Greenfield, IN, USA), intra-operative complications, and post-operative oral antibiotic therapy. All preoperative and postoperative TPA’s were measured by one author (YA). Available pre and recheck follow up radiographs were interpreted by a board-certified radiologist (YH) and OA scores were assigned to each radiograph. Each radiograph was assigned two different OA scores based on previously published literature [32, 33]. Data collected from follow up visits included: appearance of the incision or any short-term complications at the 2 week recheck/suture removal examination, lameness score at the time of suture removal and at the time of recheck radiographs (on average 6–10 weeks postoperatively), any long-term complications (up to 2 years following the procedure), and whether a bilateral staged TPLO was performed during the study period. Short term complications were considered those occurring from time of surgery to suture removal and long-term complications were considered those occurring from suture removal to the last available follow up. Complications included: signs consistent with surgical site infection (SSI) during the entire follow up period, need for implant removal, persistent lameness, or any implant complications or procedure related fractures. Surgical procedures All cases were anesthetized using protocols determined by the primary clinician or board-certified anesthesiologist which included premedication with hydromorphone 0.1mg/kg IV, induction with propofol titrated to effect 6mg/kg IV, and isoflurane maintenance. Patients also received cefazolin 22mg/kg IV every 90 minutes for the duration of surgery and then every 8 hours for 24 hours post-operatively. All dogs underwent TPLO by a board-certified small animal surgeon or a surgery resident under direct supervision of a board-certified surgeon. The decision to use lPRP was based on surgeon’s preference and owner financial ability. Meniscal debridement via caudal pole hemimeniscectomy was performed only if a meniscal tear was diagnosed. Meniscal release was not performed in any of the groups. LPRP was administered upon closure of the arthrotomy, and the volume recorded. All plates were Depuy Synthes locking TPLO plates (Synthes, Philadelphia, USA). Post-operative care, hospitalization and discharge All cases were hospitalized for at least 24 hours following surgery. Most cases were discharged within 24 hours from the time of surgery, and if they stayed longer, it was due to owner preference. Unless contraindicated, all dogs were discharged with appropriate dose of non-steroidal anti-inflammatory medications for 10 days and gabapentin 5–10 mg/kg orally every 8–12 hours for 7–14 days. All incisions were covered with an adherent covering (Primapore, Smith+New, Watford, England, UK) while in hospital. At the time of discharge, no patient had an incision cover or bandage in place. Some cases from both groups were discharged on a 7-day course of oral antibiotics (Cefpodoxime 5–10 mg/kg PO once daily) based on surgeon preference. Leukoreduced PRP preparation LPRP (ACP) was prepared following previously published guidelines [34, 35]. Briefly, once dogs were premedicated for surgery, blood was obtained using aseptic technique with either an 18-gauge butterfly catheter placed in the jugular vein or from cephalic vein during intravenous catheter (IVC) placement. Approximately 10-15mls of blood was collected in the provided double syringe mechanism (Arthrex incorporations, Naples, Florida). The blood was centrifuged at 1500 revolutions per minute (rpm) for 5 minutes (Hettich Rotofix 32, Arthrex Inc.). Centrifugation separates the red blood cells and majority of the white blood cells into a separate compartment of the syringe from the plasma. Total lPRP preparation time was approximately 20 minutes. With this system, no anticoagulant citrate is used. lPRP injection & treatment The lPRP was transferred into a sterile syringe intra-operatively and injected into the stifle joint (1.5-3ml depending on the amount available) upon closing the arthrotomy. The remaining lPRP (approximately 0.5-1ml) was applied over the osteotomy, and TPLO plate after copious lavage and prior to closure of the fascial layer. Recheck examination & suture removal Approximately 10–14 days post-surgery, all cases returned for a recheck examination and suture removal. Appearance of the incision, lameness score, any short-term complications (those occurring from surgery to the suture removal), and any evidence of surgical site infection, were documented. If any incisions showed evidence of purulent discharge concerning for incision infection, an aerobic culture and sensitivity was recommended. Recheck radiographs A recheck exam with sedated radiographs to assess osteotomy healing was recommended 6–10 weeks post-surgery (unless there were any complications in which case, patients were evaluated earlier). Lameness score, appearance of the incision, long term complications (those occurring from suture removal to last available follow up), radiographic evidence of osteotomy healing, stability of the stifle under sedation and any other complications were documented. Radiographic OA score All available pre-operative and follow up recheck radiographs were assessed by a single board-certified radiologist (YH) and two different OA scores assigned to each radiograph (as defined below). The radiologist was blinded to the groups. The OA scores were selected to provide two different OA scores for each radiograph [32, 33]. The objective OA scoring system (32) consisted of: Grade 0 = normal/no OA, effusion or osteophytes, grade 1 = early OA, stifle effusion only; no osteophytes present, grade 2 = mild OA, osteophytes on patella and femoral trochlea ridges only, grade 3 = moderate OA, small osteophytes on patella, femoral trochlea ridges, femoral condyles, fabellae, periarticular margins of the tibial plateau, and fibular head only, grade 4 = moderate to severe OA, medium to large osteophytes on patella, femoral trochlea ridges, femoral condyles, fabellae, periarticular margins of the tibial plateau, and fibular head only; mild to moderate subchondral sclerosis, and grade 5 = severe OA, osteophytes on patella, femoral trochlear ridges, femoral condyles, fabellae, periarticular margins of the tibial plateau, fibular head, and within the intercondylar notch; marked calcification and subchondral sclerosis. The subjective OA score [33] describes the following: Global score for overall disease severity (grade 0 to 3). The global score was a summation of these categories: joint effusion (grade 0 to 2), osteophytosis (grade 0 to 3), intra-articular mineralization (grade 0 to 2) and tibial subchondral sclerosis (grade 0 to 1). Surgical site infection (SSI), antibiotic therapy and implant removal The following physical examination findings were considered consistent with a surgical site infection based on previously published guidelines [36, 37]: a) Purulent drainage from the incision, b) Bacteria aseptically cultured from the incision site, c) Presence of a draining tract, d) Heat, redness, pain, or localized swelling) Incision reopened by surgeon unless there were negative culture results. Redness, inflammation and swelling that did not lead to incision opening and a positive culture, was not considered as surgical site infection, and was categorized as inflamed incision. The primary investigator (YA) reviewed all records and determined categorization for clinical SSI or inflamed. Any implant removal was recorded, and any culture result documented. Dogs from either group that were prescribed oral antibiotics at discharge, were excluded from this part of the study. Statistical analysis Descriptive statistics Data was assessed for normality with a Shapiro- Wilk test. Descriptive statistics were reported as frequency for each category (percentage), mean +/- standard deviation (SD) for normally distributed data. Medians were used where data was not normally distributed. All counted values except mean pre-op and post-op TPA were done using binomial tests (against an even split of 50%-50%). The mean pre-op and post-op TPA range was done using a two-sample t-test. Determining differences between PRP and non-PRP groups To ensure that the PRP and non-PRP groups were comparable, tests were conducted on various attributes. To determine that groups were not significantly different with regards to gender or the presence of a meniscal tear, chi-square tests were run. To determine that the groups were not significantly different with regards to age or weight, t-tests were run. To determine with there was no significant difference with regards to BCS, a Fisher’s exact test was run. Modeling effects of PRP To investigate the effects of PRP and other variables on the outcomes of interest, regression models were created. Because some dogs were represented multiple times within these data, multi-level regression models were created using patient as a random effect. For the dichotomous outcome variables of worsening of OA (for all aspects of OA assessment such as Effusion score, Global score, osteophyte score), multi-level logistic regression was used. Predictors of PRP group, gender, age, weight, BCS, and presence of a meniscal tear were included as potential variables in the model to determine which were significant. To compare the lengths of hospital stays, a linear mixed model was created which included the same list of potential predictors as the logistic models above. To compare effusion change scores, a multinomial regression model was created where the outcome variable was categorized as being better, worse, or the same. To avoid assumptions about the relationships between these three levels and the predictors, effusion change was not considered to be an ordered variable. The code and data are posted to https://github.com/stats-matt/PRP-study. Results Signalment data A total of 110 cases met the study inclusion criteria with 54 TPLOs in the lPRP group and 56 in the control (C) group. The median age for all cases was 6 years old (1–13 years). The median age for the C group was 6 years (1–13 years) and the median age for the lPRP group was also 6 years old (2–11 years). The population of the dogs that received lPRP and those that did not receive lPRP were not significantly different. There were no significant differences in these populations with regard to gender (p = 0.17), age (p = 0.59), presence of meniscal tear (p = 0.39), weight (p = 0.06), or BCS (p = 0.67). Forty-seven dogs were castrated male, 60 were female spayed, two male intact and one female intact. Dogs were of a variety of breeds with Pitbull being the most common breed (35 cases). Other represented breeds included: German shepherds (12), Labrador Retriever (12), English bulldog (8), mix breed (7), golden retriever (7), Boxer (4), Alaskan Malamute (3), American Staffordshire terrier (3), husky (2), Newfoundland (2), Rottweiler (2), sheepdog (2), Akita (2), Doberman Pincher (2), Mastiff (1), cocker spaniel (1), standard poodle (1), pointer (1), and whippet (1). Mean body weight for all dogs was 29.2 kilograms (kg). Mean body weight was 28.3 and 29.7 kg for C and lPRP group, respectively. Mean body condition score (BCS) was 5 (out of 9) for all cases. Mean BCS for each group was 5 out of 9. Surgical data (Table 1) 10.1371/journal.pone.0287922.t001 Table 1 Summary of surgical data for all cases. Variable Overall Control PRP P value Right TPLO 45 22 23 1.00 Left TPLO 33 14 19 0.487 Bilateral staged TPLO 16 10 6 0.454 Mean Pre-op TPA (range) 28.3 (23–45) 27.8 (23–32) 28.7 (23–45) 0.077 Mean post-op TPA (range) 6.9 (1–13) 7.7 (1–13) 6.2 (1–13) 0.003 Meniscal tear 43 19 24 0.542 Liposomal bupivacaine injection 60 40 20 0.013 Intra-op synovitis (reported) 69 47 22 0.004 Mild 39 32 7 <0.001 Moderate 26 11 15 0.557 Severe 4 4 0 0.125 Intra-op OA (reported) 53 30 23 0.41 Mild 15 8 7 1.00 Moderate 34 19 15 0.608 Severe 4 3 1 0.625 TPLO plate type 110 56 54 0.924 3.5mm mini 21 16 5 0.027 3.5mm standard 69 34 35 1.00 3.5mm broad 19 5 14 0.064 2.7mm 1 1 0 1.00 Intra-op complication 10 3 7 0.344 The results of TPLO leg, pre-op platelet count, pre-op TPA, post-op TPA, meniscal status, Liposomal Bupivacaine injection, intra-op synovitis, intra-op OA assessment, TPLO plate type and intra-op complications are summarized in Table 1. Briefly, 45 cases had right TPLO, 33 had left TPLO, and 16 cases underwent bilateral staged TPLO between both groups. In the C group, 22 cases had right TPLO, 14 had left TPLO, and 10 cases had bilateral staged TPLO. In the lPRP group, 23 cases had right TPLO, 19 cases had left TPLO, and 6 dogs had bilateral staged TPLO. The mean preoperative platelet count for the lPRP group was within normal reference range at 236. 9k/μL (SD = 59.4). Mean pre-operative TPA for all cases was 28.3 degrees. Mean pre-operative TPA were 27.8 and 28.7 degrees in C and lPRP group, respectively. There was no significant difference in TPA between groups (Two sample t-test, p = 0.077). Mean post-op TPA was 7.68 (SD = 2.75) and 6.18 (SD = 2.27) for the C and lPRP groups respectively (p = 0.003). Sixty cases received local injection of bupivacaine liposome injectable suspension at 0.4 ml/kg and 50 cases did not. In the C group 40 cases received liposomal bupivacaine injection upon closure of the fascia while 16 cases did not. In the lPRP group, 20 cases received liposomal bupivacaine injection and 34 cases did not. Among the 110 cases, 66 had intact meniscus, while 43 were diagnosed with a medial meniscal tear. One surgery report did not comment on the status of the meniscus. In the C group, 36 menisci were diagnosed to be intact while 19 medial meniscal tears were diagnosed (one did not comment on the status of the meniscus). In the lPPR group, 30 menisci were intact and 24 were diagnosed with medial meniscus tear. Subjective intra-operative synovitis and OA were also documented in each group. Overall, intra-operative synovitis was documented in 69 surgery reports, with 39 cases being mild, 26 moderate and 4 severe synovitis. In the C group, 32 cases had mild intra-op synovitis, 11 had moderate synovitis, and 4 cases had severe intra-op synovitis. In the PRP group, 7 had mild synovitis, 15 had moderate synovitis and no case of severe intra-operative synovitis was noted. Statistically there was a significant difference in intraoperative assessment of synovitis, with more cases with synovitis reported in the control group compared to the PRP group. Intra-operative OA was documented in 53 cases overall with 15 being mild, 34 being moderate and 4 being severe. In the C group, 8 cases had mild OA, 19 had moderate and 3 had severe. In the PRP group, 7 had mild, 15 had moderate and 1 case had severe OA. Surgical site infection, incision appearance, lameness scores, complications at suture removal, and plate removal rate Within the 110 cases recruited in the study, 31 cases were prescribed antibiotics at discharge. These cases were excluded from the infection-related data analysis. Out of 79 cases that were not discharged with oral antibiotics, 42 were in the C group while 37 were in the lPRP group. The results are summarized in Table 2. 10.1371/journal.pone.0287922.t002 Table 2 Summary of post-operative lameness score assessment, osteotomy healing, complications and SSI. Variable Overall Control PRP P value Lameness score (0–4) at suture removal 108 55 53 0.923 0 1 0 1 1.00 1 51 18 33 0.049 2 48 33 15 0.013 3 7 4 3 1.00 4 1 0 1 1.00 Lameness score (0–4) at recheck radiographs 93 46 47 1.00 0 63 26 37 0.207 1 25 17 8 0.108 2 4 3 1 0.625 3 1 0 1 1.00 4 0 0 0 NA Incision appearance at suture removal Infected 4 3 1 0.625 Inflamed 14 9 5 0.424 Swollen 18 12 6 0.238 Did not comment 2 1 1 1.00 Normal 72 31 41 0.289 Complication at suture removal 16 8 8 1.00 Complication at recheck radiographs 26 13 13 1.00 SSI 16 10 6 0.455 TPLO explant surgery 8 2 6 0.289 Among these cases at suture removal appointment, 4 incisions appeared infected, 14 incisions looked inflamed, 18 incisions were swollen, two had no comment on the incision appearance and 72 incisions had healed normally. The P-values are reported in Table 2. Overall, 16 SSIs were diagnosed from the time of suture removal until the last available follow up (at least 6 months post-surgery). In the C group, 10 cases developed surgical site infection and in the lPRP group 6 cases developed surgical site infection. Implant removal surgery was recommended for all cases. Two cases in the C group underwent TPLO implant removal and 6 cases in the lPRP group had the TPLO implants removed. The remaining cases did not undergo implant removal due to financial reasons or owners deciding against putting their pet through another procedure. Osteoarthritis and osteotomy healing The results of pre-operative OA scores, radiographic healing of the osteotomy and comparison between the groups are summarized in Table 3. 10.1371/journal.pone.0287922.t003 Table 3 Summary of post-operative OA scores and radiographic evidence of osteotomy healing. Variable Overall Control PRP P value Mean pre-op OA score (range 0 to 5) 2.4 2.25 2.6 0.049 Mean post-op OA score (range 0 to 5) 2.7 2.6 2.8 0.381 Mean pre-op global OA score (range 0 to 3) 1.25 1.25 1.3 0.796 Mean post-op global OA score (range 0 to 3) 1.3 1.4 1.25 0.176 Mean pre-op effusion OA score (range 0 to 2) 1.7 1.6 1.8 0.103 Mean post-op effusion OA score (range 0 to 2) 1.6 1.5 1.7 0.163 Mean pre-op osteophytosis OA score (range 0 to 3) 1.2 1.1 1.28 0.107 Mean post-op osteophytosis OA score (range 0 to 3) 1.3 1.3 1.3 0.976 Mean pre-op mineralization OA score (range 0 to 2) 0.3 0.4 0.25 0.129 Mean post-op mineralization OA score (range 0 to 2) 0.45 0.6 0.3 0.026 Osteotomy healing at recheck radiographs 62 22 40 0.03 Changes in OA score were classified as either progressive or not progressive. If the global OA score increased after the surgery, the dog was considered to have progressive OA. If the score stayed the same, it was considered not progressive. If the post-surgery score was decreased compared to the pre-surgery score, the dog was considered not progressive. The number of dogs with progressive OA in the C group (4) was twice that of the lPRP group (2). The degree of changes in the OA scores between pre and post radiographs was compared between the groups and p-values reported in Table 3. There was no statistical difference in OA score changes between the groups (p = 0.072). For the degree of global OA score change comparison between the C and lPRP group, the lPRP group had significantly less progression of their global OA scores compared to the C group when pre- and post-op global OA scores were considered (p = 0.033). A line graph showing the average change in outcomes of global OA for dogs with and without lPRP is depicted in Fig 1. 10.1371/journal.pone.0287922.g001 Fig 1 Line graph showing the predicted average change in outcomes of global OA for each of the PRP and non-PRP groups. N = not administered PRP, Y = administered PRP. Modeling change in osteophytosis scores between pre-op and post-op and comparison between groups A total of 10 dogs had a progressive osteophytosis score (OS score) on post-op radiographs compared to their pre-op: 6 among the C and 4 among the lPRP group. While there was no significant difference in pre and post-op OS score progression between the two groups (p = 0.277), there was a significant difference between pre and post-op OS score progression between dogs that had an intact medial meniscus, compared to dogs that had a medial meniscal tear (p = 0.024). Modeling change in pre-op and post-op mineralization scores and comparison between groups The degree of mineralization score change (comparing pre-op to post-op mineralization scores) was not significantly different between the two groups (p = 0.659) though the probability of mineralization progressing in the lPRP group was lower compared to the C group (p = 0.18). Degree of change for effusion scores and comparison between groups For dogs in the C group, 66% of the effusion scores stayed the same without any progression while 23% had less effusion score (lower numbers) and 11% had increased effusion scores (higher numbers). For dogs in the lPRP group, the vast majority (80%) stayed the same and there was no progression of effusion score, while 10% had decreased effusion score and the other 10% had increased effusion scores. This difference between groups was not statistically significant (p = 0.225). Radiographic evidence of osteotomy healing Radiographs were assessed 6–10 weeks post-surgery (on average 8 weeks). There was no significant difference in the timing of assessment between treatment groups. Overall, 62 cases had evidence of osteotomy healing at the time of recheck radiographs (assessed by board certified radiologist). In the C group, 22 cases had evidence of osteotomy healing while 40 cases in the PRP group had evidence of complete osteotomy healing. Radiographic evidence of osteotomy healing was compared between the two groups: Both PRP and age were significant predictors of osteotomy healing (p = 0.003 and p = 0.049, respectively.) For the average aged dog (5.84 years), the probability of radiographic healing for dogs in the C group was 51.1% compared to dogs in the lPRP group which was 87.6%. This indicates that as age increases, the probability of healing decreases, but for all ages, the probability of healing is higher for dogs in the lPRP group compared to dogs in the C group. The relationship between healing, age, and PRP can be seen in Fig 2. 10.1371/journal.pone.0287922.g002 Fig 2 Scatterplot with curves represent probability of osteotomy healing by age for each of the PRP and non-PRP groups. N = not administered PRP, Y = administered PRP. SSI There is no indication for a relationship between risk of infection in the lPRP group compared to C group. (P≤0.5, one tailed proportion test). Dogs from either group that were discharged on oral antibiotics were excluded from this part of data analysis. If the cases that were discharged on antibiotics were to be included in the study, then there would be significantly less infections in the lPRP group compared to C group (p = 0.012, one tailed proportion test). Lameness score at recheck exam and comparison between groups When comparing the change in lameness score at from suture removal to score at recheck radiographs examination (6–10 weeks post-operative), dogs in the lPRP group had significantly lower lameness scores compared to dogs in the C group (p = 0.003). Having received PRP treatment, resulted in a decrease of lameness score by 0.412 on average when comparing pre-op and post-op lameness scores in dogs of the lPRP group. Discussion The purpose of this study was to evaluate the effects of concurrent PRP injection at the time of TPLO and compare SSI rate, implant removal rate, degree of change in OA progression score, lameness score progression and radiographic bone healing. We found that PRP treatment significantly improved radiographic healing of the osteotomy, improved global OA scores and improved lameness score at recheck examination. We found no significant difference between the lPRP and C group with regard to SSI and implant removal rate. Our groups in this study were homogenous with regards to age, weight, breed, pre-op TPA, the affected limb and meniscus status at the time of surgery. Postoperative TPA was found to be significantly different between groups which could, in theory, have affected postoperative outcomes such as lameness score and possibly osteoarthritis scores as the lPRP group TPA was closer to the published preferred angle of 5º. While statistically significant, the mean difference was only 1.5º, which is within the intraobserver error published previously [38] and the authors feel this difference is likely to be clinically insignificant. Even though there was a significant difference in intraoperative assessment of synovitis, with more mild synovitis in the control group compared to the PRP group this grading score was a subjective assessment, therefor this finding is unlikely to be of clinical importance. Future studies could evaluate the postoperative outcomes of lPRP compared to more objective intraoperative synovial biopsies. We rejected our hypothesis that PRP would have no effect on osteotomy healing. This contrasted with the findings of Franklin et al. [29] where PRP administration did not improve proximal tibial osteotomy healing. This finding could be due to multiple factors. Firstly, our larger case numbers could have played a role (54 dogs per group in our study compared to 27 per group in Franklin’s study). Secondly, criteria for healing were different between the two studies. We evaluated osteotomy healing solely on plain radiographs, whereas plain radiography, ultrasound and MRI were evaluated in the Franklin study. We evaluated radiographic osteotomy healing on average 8.34 weeks post-surgically, while radiographic osteotomy healing was assessed at 28, 49, and 70 days in the Franklin study. This could affect the documented osteotomy healing, as radiographic healing is expected to progress as more time elapses. The results of our study reveal that radiographic healing at approximately 8 weeks post-surgery was significantly better in the PRP compared to C group. Both studies found age to be a significant factor affecting osteotomy healing time concluding as age increases, a longer time to radiographic osteotomy healing should be expected. Other studies have evaluated the effects of PRP on bone healing in canine patients. Rabillard et al. [39] looked at the effects of autologous platelet rich plasma gel and calcium phosphate biomaterials on bone healing in an ulnar ostectomy model in dogs and found no improved bone healing with PRP. Souza et. al. on the other hand, found PRP to be effective in promoting bone healing in a canine radial ostectomy gap model [40]. Gianakos et. al. performed the most robust systemic review to date on the effects of PRP in animal long bone model [21] and found that 89% of studies reported significant improvement in earlier bone healing on histologic assessment. One hundred percent of patients showed significant increase in bone formation on radiographs with the PRP and 100% showed a higher torsional stiffness for the PRP-treated defects. They concluded that PRP is beneficial as a biologic adjunct in animal long-bone models. The results of the latter two studies are consistent with the results of our study. We employed two different OA scoring systems for this study to be as objective as possible, and to compare results using two validated systems [32, 33]. The results illustrate a lower rate of OA global score progression in cases that received lPRP compared to the C group. This is consistent with multiple other studies that showed improvement in OA with PRP administration [19, 22, 23, 27, 34, 41, 42]. These results should be interpreted with caution, due to the lack of long-term radiographic follow up however 6 months follow up is consistent with some studies [22, 42]. The authors speculate whether the global OA scores would have remained significantly in favor of PRP if long term radiographs were assessed, for example,12 or 24 months post-operatively, since OA progression is expected to slow down following stabilization of CCL deficiency, regardless of the use of PRP [4–6]. The results of our study illustrated an improved lameness score on 6–10 week recheck examination for dogs following TPLO in the lPRP group compared to dogs in the C group. The results should be interpreted with caution as clinical lameness score is subjective and varies from surgeon to surgeon. The surgeons in this study routinely used this grading system and studies have shown that intraobserver variations are within acceptable limits. One theory for improved lameness score in lPRP group could be faster healing of the osteotomy leading to earlier load bearing and improved lameness score. The lPRP is supposed to decrease inflammation through multiple pathways and this could be the primary mechanism behind the improved lameness scores as well. Future studies using an objective means of lameness assessment such as a force plate analysis are warranted. The incidence of SSI was not significantly different between groups, which rejects our hypothesis. There are published studies highlighting the antibacterial effect of PRP in wounds [16]. Although other studies have shown antibacterial effects of PRP on methicillin resistant wounds in dogs, our study failed to show any significant difference in implant infection rate between the lPRP and the control group after removing the cases that were discharged on oral antibiotics. Although the number of dogs in each group was small after removing the patients that were treated with post operative antibiotics: ten dogs in the C group developed SSI (4 were excluded because they were discharged with oral antibiotics), and 6 dogs in the lPPR group developed SSI (0 were discharged with oral antibiotics). The authors speculate that the insignificant results could be due to low number of cases in each group (Type II error) or differences in lPRP centrifugation methodology. Interestingly, when dogs that received oral antibiotics were not excluded from this portion of the study, the results would have shown a statistical significance for reduced rate of SSI in the lPRP group. There is conflicting evidence in the literature on the role of post-operative oral antibiotic therapy in reducing the rate of SSI following TPLO. The most recent systematic literature review study failed to find an association between post-operative antibiotic therapy and decreased risk of SSI following TPLO in dogs [13]. Implant removal rate was also not significantly different between the 2 groups which rejects our hypothesis. This is most likely due to the low number of cases that required implant removal in both groups and the fact that the recommendation to remove the TPLO plate was not pursued by all owners for financial reasons. It is also possible that some cases were lost to follow up, since implant infection can occur even years after the initial procedure. This study has several limitations inherent to a retrospective study. Cases were excluded due to incomplete medical records, and all follow-up examinations were based on the available medical records at our hospital or records that we received from the referring veterinarians. Financial limitations of clients were the primary reason to not pursue implant removal for the cases that developed SSI, which affected our implant removal case numbers. Follow-up time was also not strictly controlled, as in most retrospectives. Some of the earlier cases had up to 48 months follow up, while the more recent cases had only 6 months of follow up. In conclusion, concurrent intra-articular injection and plate surface treatment with leukocyte reduced PRP at the time of TPLO, is beneficial in slowing the progression of OA (compared to the control group), hastening the radiographic evidence of osteotomy healing, and improved lameness score on recheck examination. LPRP was not a significant factor in reducing SSI or implant removal rate. The authors would like to acknowledge Matt Thomas, PhD, for his work on the statistical analysis for this report. 10.1371/journal.pone.0287922.r001 Decision Letter 0 Abdel-Wanis Mohamed El-Sayed Academic Editor © 2023 Mohamed El-Sayed Abdel-Wanis 2023 Mohamed El-Sayed Abdel-Wanis https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version0 5 Apr 2023 PONE-D-22-35213Multifactorial assessment of leukocyte reduced platelet rich plasma injection in dogs undergoing tibial plateau leveling osteotomy: a retrospective studyPLOS ONE Dear Dr. Aryazand Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by May 20 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Mohamed El-Sayed Abdel-Wanis, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at  https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and  https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ********** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ********** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ********** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The paper is very interesting and well written. As a work on PRP, the authors may consider including data on the number of platelets in the blood of patients before surgery, which, with the centrifugation method used, would allow for the standardization of the PRP used. Reviewer #2: This manuscript examined the effects of leukocyte reduced platelet rich plasma (lPRP) on surgical site infection (SSI), implant removal, bone healing, clinical signs, progression of OA in the dogs undergoing tibial plateau leveling osteotomy (TPLO). As the results, lPRP treatment significantly improved bone healing, global OA scores, and lameness score. However, there were no significant differences in the results of SSI and implant removal rate. The content of this paper is worthy of publication in Plos One, because of its contribution to the clinical field of veterinary medicine. However, some revisions of manuscript are needed before it can be accepted for publication. Introduction (line 79): “1PRP” is a first appearance. Please list the term that is not abbreviations (Ex. Leukoreduced platelet rich plasma?). M&M (line 87-93, 136-137): How did you divide the subject dogs into two groups? What were the criteria of surgeon's preference? M&M (line 101-103): Orthopedic (excluding CrCL rupture) and neurological disorders that may affect gait and mobility should be also added to the exclusion criteria. M&M (line 139-142): This content is described below (lines 164-167), so delete the relevant text. M&M (line 150): pet→dog M&M (line 151): Please describe which type of antibiotic was administered orally. Results (line 262-270): Did you compare post-op TPA between groups? These values might affecte the outcome of the surgery. Results (line 277-281): Add the sentence about significant difference in the presence of synovitis between groups. Table 2: Correct any misalignments in the Table 2 (Incision appearance at suture removal). Discussion (line 415-425): In this paragraph, you need to add a discussion of the role of PRPs in infection protection and the results of this study. Discussion (line 438-442): leukocyte reduced PRP→lPRP? Discussion: In this study, lameness was significantly improved in the lPRP group. You should discuss whether the accelerated bone healing in the lPRP group was the result of improved loading on the tibial bone or the effect of PRP in the Discussion. Reference: There are two No. 29 references. Please check again, including the citation in the text. ********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes: Kazuya Edamura ********** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. 10.1371/journal.pone.0287922.r002 Author response to Decision Letter 0 Submission Version1 21 May 2023 Response to Reviewers Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The paper is very interesting and well written. Author response: Thank you. We appreciate your support and the time you have taken to review our manuscript. As a work on PRP, the authors may consider including data on the number of platelets in the blood of patients before surgery, which, with the centrifugation method used, would allow for the standardization of the PRP used. Author response: Thank you for bringing up this interesting point. We have added this information to the methods and results section for the lPRP group. We did not feel statistical analysis was necessary for the platelet count as there is no gold standard number to compare to and the data was normally distributed. Please let us know if you feel we need to add this in more detail. Reviewer #2: This manuscript examined the effects of leukocyte reduced platelet rich plasma (lPRP) on surgical site infection (SSI), implant removal, bone healing, clinical signs, progression of OA in the dogs undergoing tibial plateau leveling osteotomy (TPLO). As the results, lPRP treatment significantly improved bone healing, global OA scores, and lameness score. However, there were no significant differences in the results of SSI and implant removal rate. The content of this paper is worthy of publication in Plos One, because of its contribution to the clinical field of veterinary medicine. However, some revisions of manuscript are needed before it can be accepted for publication. Author response: Thank you for your time and your suggestions. We have tried to address each of your concerns below. Introduction (line 79): “1PRP” is a first appearance. Please list the term that is not abbreviations (Ex. Leukoreduced platelet rich plasma?). Author response: The first appearance of the term lPRP is actually in line 15 where the full term (Leukoreduced Platelet rich Plasma) has been listed. We have added it to line 79 as well just to be safe. M&M (line 87-93, 136-137): How did you divide the subject dogs into two groups? What were the criteria of surgeon's preference? Author’s Response: The dogs were divided into groups based on surgeon’s preference. One surgeon (NJB) and one surgery resident (YA) preferred patients receive lPRP injection while other surgeons and residents did not. Not all cases of NJB or YA received lPRP though as sometimes the owners declined the cost of lPRP injection, thus those cases had routine TPLO without PRP injection. We have added this information into the methods section in both of the places you referenced. M&M (line 101-103): Orthopedic (excluding CrCL rupture) and neurological disorders that may affect gait and mobility should be also added to the exclusion criteria. Author response: Thank you for this suggestion. We have added this to the exclusion criteria. M&M (line 139-142): This content is described below (lines 164-167), so delete the relevant text. Author Response: Thank you for your comment. We have deleted the redundant content (lines 139-142) per your suggestion. M&M (line 150): pet→dog Author response: Changed pet to patient. M&M (line 151): Please describe which type of antibiotic was administered orally. Author response: Thank you for your comment. We have added the antibiotic name and dose. Cefpodoxime (simplicef) 5-10 mg/kg PO once daily Results (line 262-270): Did you compare post-op TPA between groups? These values might affect the outcome of the surgery. Author response: Thank you for this suggestion. We did not originally compare postoperative TPAs but have done so as we agree this might affect some of the postoperative outcomes (possibly lameness score and osteoarthritis score) we looked at. When analyzed there was a statistically significant difference between lprp and control groups however this mean difference was only 1.5º which is within the published intraoberver difference for TPA (added reference 38). We have added this information and stated we do not feel such a small TPA difference would have an effect clinically. Results (line 277-281): Add the sentence about significant difference in the presence of synovitis between groups. Author Response: Thank you for this comment. We have added a line in the results as well as the discussion. We feel the subjectivity of this score limits its clinical value but feel like future works could evaluate outcomes based on more rigorous operative diagnostics such as biopsies. Table 2: Correct any misalignments in the Table 2 (Incision appearance at suture removal). Author response: Thank you for catching this. We have corrected the misalignment. Discussion (line 415-425): In this paragraph, you need to add a discussion of the role of PRPs in infection protection and the results of this study. Author response: We have added information in this section. Please let us know if you feel this needs continued supplementation. Discussion (line 438-442): leukocyte reduced PRP→lPRP? Author response: Changed Discussion: In this study, lameness was significantly improved in the lPRP group. You should discuss whether the accelerated bone healing in the lPRP group was the result of improved loading on the tibial bone or the effect of PRP in the Discussion. Author response: Thank you for this comment. We have added a few sentences to the discussion regarding the differences in lameness scores. Reference: There are two No. 29 references. Please check again, including the citation in the text. Author response: Changed and adjusted all the references in the text accordingly. Attachment Submitted filename: Response to PLoS One reviewers 5-20-23.docx Click here for additional data file. 10.1371/journal.pone.0287922.r003 Decision Letter 1 Abdel-Wanis Mohamed El-Sayed Academic Editor © 2023 Mohamed El-Sayed Abdel-Wanis 2023 Mohamed El-Sayed Abdel-Wanis https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version1 15 Jun 2023 Multifactorial assessment of leukocyte reduced platelet rich plasma injection in dogs undergoing tibial plateau leveling osteotomy: a retrospective study PONE-D-22-35213R1 Dear Dr. Nicole Buote We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Mohamed El-Sayed Abdel-Wanis, Ph.D. Academic Editor PLOS ONE 10.1371/journal.pone.0287922.r004 Acceptance letter Abdel-Wanis Mohamed El-Sayed Academic Editor © 2023 Mohamed El-Sayed Abdel-Wanis 2023 Mohamed El-Sayed Abdel-Wanis https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 22 Jun 2023 PONE-D-22-35213R1 Multifactorial assessment of leukocyte reduced platelet rich plasma injection in dogs undergoing tibial plateau leveling osteotomy: a retrospective study Dear Dr. Buote: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Dr Mohamed El-Sayed Abdel-Wanis Academic Editor PLOS ONE ==== Refs References 1 Wilke VL , Robinson DA , Evans RB , Rothschild MF , Conzemius MG . Estimate of the annual economic impact of treatment of cranial cruciate ligament injury in dogs in the United States. J Am Vet Med Assoc. 2005;227 : 1604–1607. doi: 10.2460/javma.2005.227.1604 16313037 2 Nicoll C , Singh A , Weese JS . Economic impact of tibial plateau leveling osteotomy surgical site infection in dogs. Vet Surg. 2014;42 : 899–902. 3 Nanda A , Hans EC . Tibial Plateau Leveling Osteotomy for Cranial Cruciate Ligament Rupture in Canines: Patient Selection and Reported Outcomes. Vet Med (Auckl). 2019;10 : 249–255.31921614 4 Hurley CR , Hammer DL , Shott S . Progression of radiographic evidence of osteoarthritis following tibial plateau leveling osteotomy in dogs with cranial cruciate ligament rupture: 295 cases (2001–2005). J Am Vet Med Assoc. 2007 Jun 1;230 (11 ):1674–9. doi: 10.2460/javma.230.11.1674 17542736 5 Shimada M , Mizokami N , Ichinohe T , Kanno N , Suzuki S , Yogo T , et. al . Long-term outcome and progression of osteoarthritis in uncomplicated cases of cranial cruciate ligament rupture treated by tibial plateau leveling osteotomy in dogs. J Vet Med Sci. 2020;82 : 908–916.32448811 6 Gatineau M , Dupuis J , Plante J , Moreau M . Retrospective study of 476 tibial plateau leveling osteotomy procedures. Vet Comp Orthop Traumatol. 2011;24 : 1–9.20830454 7 Innes JF , Bacon D , Lynch C , Pollard A . Long-term outcome of surgery for dogs with cranial cruciate ligament deficiency. Vet Rec. 2000;147 : 325–328.11058021 8 Fitzpatrick N , Solano MA . Predictive variables for complications after TPLO with stifle inspection by arthrotomy in 1000 consecutive dogs. Vet Surg. 2010;39 : 460–474.20345526 9 Frey TN , Hoelzler MG , Scavelli TD , Fulcher RP , Bastian RP . Risk factors for surgical site infection-inflammation in dogs undergoing surgery for rupture of the cranial cruciate ligament: 902 cases (2005–2006). J Am Vet Med Assoc. 2010;236 : 88–94.20043807 10 Spencer DD , Daye RM . A prospective, randomized, double-blinded, placebo controlled clinical study on postoperative antibiotic therapy in 150 arthroscopy-assisted tibial plateau leveling osteotomies in dogs. Veterinary Surgery. 2018;47 : E79–E87.30267441 11 Lopez DJ , VanDeventer GM , Krotscheck U , Aryazand Y , McConkey MJ , Hayashi K , et al . Retrospective study of factors associated with surgical site infection in dogs following tibial plateau leveling osteotomy. J Am Vet Med Assoc. 2018;253 : 315–321.30019998 12 Gallagher AD , Mertens WD . Implant removal rate from infection after tibial plateau leveling osteotomy in dogs. Vet Surg. 2012;41 : 705–711.22822724 13 Stine SL , Odum SM , Mertens WD . Protocol changes to reduce implant-associated infection rate after tibial plateau leveling osteotomy: 703 dogs, 811 TPLO (2006–2014). Vet Surg. 2018 May;47 (4 ): 481–489. doi: 10.1111/vsu.12796 29878479 14 Budsberg SC , Torres BT , Sandberg GS . Efficacy of postoperative antibiotic use after tibial plateau leveling osteotomy in dogs: A systematic review. Vet Surg. 2021 May;50 (4 ): 729–739. doi: 10.1111/vsu.13603 33709459 15 Intravia J , Allen DA , Durant TJ , McCarthy MB , Russell R , Beitzel K , et al . In vitro evaluation of the anti-bacterial effect of two preparations of platelet rich plasma compared with cefazolin and whole blood. Muscles Ligaments Tendons J. 2014;4 : 79–84.24932452 16 Farghali HA , AbdElKader NA , AbuBakr HO , Aljuaydi SH , Khattab MS , Elhelw R , et al . Antimicrobial action of autologous platelet-rich plasma on MRSA-infected skin wounds in dogs. Sci Rep. 2019 Sep 3;9 (1 ):12722. doi: 10.1038/s41598-019-48657-5 31481694 17 Kennedy MI , Whitney K , Evans T , LaPrade RF . Platelet-Rich Plasma and Cartilage Repair. Curr Rev Musculoskelet Med. 2018;11 : 573–582.30203333 18 Bozynski CC , Stannard JP , Smith P , Hanypsiak BT , Kuroki K , Stoker A , et al . Acute Management of Anterior Cruciate Ligament Injuries Using Novel Canine Models. J Knee Surg. 2016;29 : 594–603.26713594 19 Franklin SP , Garner BC , Cook JL . Characteristics of canine platelet-rich plasma prepared with five commercially available systems. Am J Vet Res. 2015;76 : 822–827.26309111 20 Malhotra A , Pelletier MH , Yu Y , Walsh WR . Can platelet-rich plasma (PRP) improve bone healing? A comparison between the theory and experimental outcomes. Archives of orthopaedic and trauma surgery. 2013;133 : 153–165. doi: 10.1007/s00402-012-1641-1 23197184 21 Gianakos A , Zambrana L , Savage-Elliott I , Lane JM , Kennedy JG . Platelet-Rich Plasma in the Animal Long-Bone Model: An Analysis of Basic Science Evidence. Orthopedics. 2015;38 : 1079–1090. 22 Kon E , Mandelbaum B , Buda R , Filardo G , Delcogliano M , Timoncini A , et al . Platelet-rich plasma intraarticular injection versus hyaluronic acid viscosupplementation as treatments for cartilage pathology: from early degeneration to osteoarthritis. Arthroscopy 2011;27 : 1490–1501.21831567 23 Filardo G , Kon E , Buda R , Timoncini A , Di Martino A , Cenacchi A , et al . Platelet-rich plasma intra-articular knee injections for the treatment of degenerative cartilage lesions and osteoarthritis. Knee Surg Sports Traumatol Arthrosc. 2011;19 : 528–535.20740273 24 Fahie MA , Ortolano GA , Guercio V , Schaffer JA , Johnston G , Au J , et al . A randomized controlled trial of the efficacy of autologous platelet therapy for the treatment of osteoarthritis in dogs. J Am Vet Med Assoc. 2013;243 : 1291–1297. doi: 10.2460/javma.243.9.1291 24134578 25 Sundman EA , Cole BJ , Fortier LA . Growth factor and catabolic cytokine concentrations are influenced by the cellular composition of platelet-rich plasma. Am J Sports Med. 2011;39 : 2135–2140.21846925 26 Oh JH , Kim W , Park KU , Roh YH . Comparison of the Cellular Composition and Cytokine-Release Kinetics of Various Platelet-Rich Plasma Preparations. Am J Sports Med. 2015;43 : 3062–3070.26473014 27 Campbell KA , Saltzman BM , Mascarenhas R , Khair MM , Verma NN , Bach BR Jr , et al . Does Intra-articular Platelet-Rich Plasma Injection Provide Clinically Superior Outcomes Compared With Other Therapies in the Treatment of Knee Osteoarthritis? A Systematic Review of Overlapping Meta-analyses. Arthroscopy. 2015;31 : 2213–21.26033459 28 Cook JL , Smith PA , Bozynski CC , Kuroki K , Cook CR , Stoker AM , et al . Multiple injections of leukoreduced platelet rich plasma reduce pain and functional impairment in a canine model of ACL and meniscal deficiency. J Orthop Res. 2016;34 :607–615.26403590 29 Franklin SP , Burke EE , Holmes SP . The effect of platelet-rich plasma on osseous healing in dogs undergoing high tibial osteotomy. PLoS One. 2017;12 : e0177597.28520812 30 German AJ , Holden SL , Moxham GL , Holmes KL , Hackett RM , Rawlings JM . A simple, reliable tool for owners to assess the body condition of their dog or cat. J Nutr. 2006;136 : 2031S–2033S.16772488 31 Hudson JT , Slater MR , Taylor L , Scott HM , Kerwin SC . Assessing repeatability and validity of a visual analogue scale questionnaire for use in assessing pain and lameness in dogs. Am J Vet Res. 2004;65 : 1634–1643.15631027 32 Moore EV , Weeren R , Paek M . Extended long-term radiographic and functional comparison of tibial plateau leveling osteotomy vs tibial tuberosity advancement for cranial cruciate ligament rupture in the dog. Vet Surg. 2020;49 : 146–154.31287180 33 Innes JF , Costello M , Barr FJ , Rudorf H , Barr ARS . Radiographic progression of osteoarthritis of the canine stifle joint: A prospective study. Vet Radiol Ultrasound. 2004;45 : 143–148.15072147 34 Smith PA . Intra-articular Autologous Conditioned Plasma Injections Provide Safe and Efficacious Treatment for Knee Osteoarthritis. An FDA-Sanctioned, Randomized, Double-blind, Placebo-controlled Clinical Trial. Am J Sports Med. 2016;44 : 884–891.26831629 35 Cross JA , Cole BJ , Spatny KP , Sundman E , Romeo AA , Nicholson GP , et al . Leukocyte-Reduced Platelet-Rich Plasma Normalizes Matrix Metabolism in Torn Human Rotator Cuff Tendons. Am J Sports Med. 2015;43 : 2898–2906. doi: 10.1177/0363546515608157 26460099 36 Horan TC , Gaynes RP , Martone WJ , Jarvis WR , Emori TG . CDC definitions of nosocomial surgical site infections, 1992: a modification of CDC definitions of surgical wound infections. Am J Infect Control. 1992;20 : 271–274. doi: 10.1016/s0196-6553(05)80201-9 1332552 37 Mangram AJ , Horan TC , Pearson ML , Silver LC , Jarvis WR . Guideline for prevention of surgical site infection, 1999. Hospital Infection Control Practices Advisory Committee. Infect Control Hosp Epidemiol. 1999;20 : 250–278.10219875 38 Fettig AA , Rand WM , Sato AF , Solani M , McCarthy RJ , Boudrieau RJ . Observer variability of tibial plateau slope measurement in 4o dogs with cranial cruciate ligament-deficient stifle joints. Vet Surg. 2003; 32 : 471–8.14569576 39 Rabillard M , Grand JG , Dalibert E , Fellah B , Gauthier O , Niebauer GW . Effects of autologous platelet rich plasma gel and calcium phosphate biomaterials on bone healing in an ulnar ostectomy model in dogs. Vet Comp Orthop Traumatol. 2009;22 : 460–466.19876529 40 Souza TFB , Andrade AL , Ferreira GTNM , Sakamoto SS , Albuquerque VB , Bonfim SRM , et al . Healing and expression of growth factors (TGF-β and PDGF) in canine radial ostectomy gap containing platelet-rich plasma. Vet Comp Orthop Traumatol. 2012;25 : 445–452.23111655 41 Sharun K , Jambagi K , Dhama K , Kumar R , Pawde AM , Amarpal . Therapeutic Potential of Platelet-Rich Plasma in Canine Medicine. Arch Razi Inst. 2021;76 : 721–730. doi: 10.22092/ari.2021.355953.1749 35096308 42 King W , Cawood K , Bookmiller M . The Use of Autologous Protein Solution (Pro-Stride®) and Leukocyte-Rich Platelet-Rich Plasma (Restigen®) in Canine Medicine. Vet Med (Auckl). 2021;12 : 53–65.33777723