Autologous Conditioned Serum Treatment Research Articles

Effects of Autologous Conditioned Serum on Non-Union After Open Reduction Internal Fixation Failure: A Case Series and Literature Review.

Background and Objectives: Non-union is a severe complication of traumatic fracture that often leads to disability and decreased quality of life, with treatment remaining complex due to a lack of standardized protocols. This study examines the effectiveness of autologous conditioned serum (ACS) for non-union in patients who have a failed open reduction internal fixation (ORIF). Materials and Methods: Eleven patients with confirmed non-union at least 9 months post-ORIF or total hip replacement were enrolled. These patients received ACS treatment on the lesion sites once to three times monthly and were followed up. Efficacy was monitored through monthly X-rays to assess callus formation and bone union. Results: Seven patients received ACS three times, three patients received it twice, and the one who underwent total hip replacement received it once. Ten patients achieved union at the last follow-up visit, indicating the effectiveness of ACS in non-union cases unresponsive to ORIF. ACS demonstrated promising results in facilitating bone union in these challenging cases. Conclusions: ACS has the potential as an alternative or adjective treatment for non-union and is worthy of being investigated further for the benefits of patients.

Effect of Application of Autologous Conditioned Serum on Wound Healing in Diabetic Mice Through Inhibition of STING Pathway Activation.

Slow wound healing in diabetic patients is a common complication of diabetes. Autologous conditioned serum (ACS) therapy is an emerging and safe biological therapy, and may accelerate the wound healing in diabetes. To investigate the effect of ACS in promoting wound healing in diabetic mice and its possible mechanism. Twenty-four six-week-old male C57BL/6J mice were selected and divided into 5 groups, including control group (Ctrl), diabetic wound group (DW), ACS treatment group (DW+ACS) and STING pathway validation group (DW+ACS+DMXAA), with six mice in each group. Intervention was initiated after the back incision was performed, and wound healing was assessed on day 0, day 7, and day 14, and wound healing was assessed by hematoxylin and eosin (HE) staining of skin tissue on day 14. At the same time, the wound healing of the fibroblast markers collagen I and α-SMA was measured by immunohistochemistry and western blot. ACS treatment significantly accelerated the diabetic wound according to the wound area and HE staining results. Meanwhile, collagen I and α-SMA concentration evaluated by immunohistochemistry and western blot were remarkably elevated under the ACS interference. The STING signaling pathway was obviously activated in diabetic wound tissues. After the addition of DMXAA, an agonist of STING, the healing function of ACS was dramatically reversed. The application of ACS promotes wound healing in diabetic mice by enhancing fibroblasts. Meanwhile, the STING signaling pathway was inactivated by ACS interference. Hence, ACS can be used in the treatment of wound healing of Diabetes.

Cytokine enrichment in equine conditioned serum is not reliant on incubation in specialized containers

Autologous conditioned serum (ACS), i.e serum enriched with anti-inflammatory cytokines and growth factors, is a popular orthobiologic therapy used in equine practice. Costly specialized tubes containing glass beads are commonly used for ACS production. The objective of this in vitro study was to compare cytokine and growth factor levels in equine serum after incubation in three different tubes: commercial plastic ACS tubes (COMM); sterile 50 ml plastic centrifugation tubes (CEN); and 10 ml plastic vacutainer tubes (VAC). Blood from 15 healthy horses was incubated in the different tubes at 37°C for 22–24 h. The concentration of IL-1β, IL-1Ra, IL-10, IGF-1 and PDGF-BB was determined by ELISA and compared between tubes. There was no difference in concentration of IL-1Ra and IGF-1 between CEN and COMM. PDGF-BB was higher in CEN vs. COMM (P < 0.0001). IL-1Ra and PDGF-BB was higher (P < 0.005 and P = 0.02, respectively) whereas IGF-1 was lower in VAC (P < 0.003) vs. the other tubes. The centrifuge tube performed similarly to the commercial ACS tube in cytokine and growth factor enrichment and has the potential to dramatically reduce the cost of ACS treatment. Cytokine enrichment of equine serum does not require blood incubation in specialized ACS containers.

Platelet Rich Plasma Versus Autologous Conditioned Serum in Osteoarthritis of the Knee: Clinical Results of a Five-Year Retrospective Study

Objectives: There is no consensus on the effectiveness of platelet-rich plasma (PRP) and autologous conditioned serum (ACS) in the treatment of knee osteoarthritis (OA). Also, the group of patients who will benefit most from this treatment is not clear. This study aims to understand the effects of two treatment modalities: ACS and PRP on pain and clinical scores in the treatment of OA. For this reason, we compared the long-term (five-year follow up) clinical results of the patients to whom these two treatment methods were applied.Materials and methods: Eighty-two knee osteoarthritis cases, selected from a database prospectively maintained in our tertiary university hospital after institutional ethics committee approval, examined between January 2013 and September 2020 and treated with ACS and PRP by the same orthobiological treatment team, were retrospectively analyzed. The clinical results of group A (n=40) treated with ACS and group B (n=42) treated with PRP were statistically analyzed. Clinical evaluations were made pre-injection and at one, six, 12, 24 and 60 months post-treatment, using the knee injury and osteoarthritis result score (KOOS) for the evaluation of function and a visual analog scale (VAS) for the evaluation of pain.Results: Side effects were noted in two patients (5%) in group A and 16 patients (38.1%) in group B. More side effects were seen in group B compared to group A (p<0.001). The better VAS scores in both groups were detected in the sixth and 12th months. When VAS scores were examined, better results were obtained in group A in the 12th and 24th months (p<0.05). When KOOS scores were examined, the superiority of ACS to PRP at 12 and 24 months was shown in KOOS.S, KOOS.P and KOOS.ADL scores (p<0.05). There was no statistically significant difference between the two groups in terms of all scores and baseline scores at 60 months.Conclusion: The effectiveness of ACS and PRP treatments can last up to two years. After two years, the effectiveness of both treatments decreases. Comparing the two treatments, ACS treatment showed better results on VAS and KOOS scores compared to PRP treatment.

Current use of biologic therapies for musculoskeletal disease: A survey of board-certified equine specialists.

ObjectiveTo evaluate the use of mesenchymal stem cells (MSCs), autologous conditioned serum (ACS), platelet‐rich plasma (PRP), and autologous protein solution (APS) for the treatment of equine musculoskeletal disease by diplomates of the American College of Veterinary Surgery (ACVS), and American College of Veterinary Sports Medicine and Rehabilitation (ACVSMR).Study designCross‐sectional study.Sample populationDiplomates (n = 423).MethodsAn email link was sent to ACVS and ACVMR diplomates. A survey contained 59 questions regarding demographics, as well as indications, frequency, adverse effects, and limitations of use. Responses were analyzed using Fisher's exact test.ResultsOne hundred and fifty four surveys were analyzed. Years in practice and type of practice were not associated with biologic therapy use. PRP was the most used therapy (120/137; 87.5%). PRP and MSCs were most often administered intralesionally while ACS and APS were most often administered intra‐articularly. ACS (50/104; 48.1%) treatment was repeated commonly within 2 weeks of initial injection. MSCs (39/90; 43.3%) and PRP (38/100; 38%) were commonly repeated 1‐2 months after initial injection and APS was typically repeated >4 months after initial injection (21/53; 39.6%). Local inflammation and expense were the most common adverse effect and limitation of use.ConclusionDiplomates most commonly utilized PRP and MSC intralesionally for soft‐tissue injuries, and ACS and ACP intra‐articularly for joint injury. Protocols for repeated administration varied widely. Local inflammation was a clinical concern with the use of biologics.Clinical significanceBiologic therapies are used commonly by ACVS and ACVSMR diplomates for soft tissue and joint disease.

Use of autologous conditioned serum dressings in hard-to-heal wounds: a randomised prospective clinical trial.

In this study, we aimed to assess both the efficacy and tolerability of autologous conditioned serum (ACS) as an innovative wound dressing in the local management of hard-to-heal wounds. In this single-blinded randomised controlled trial, patients with hard-to-heal wounds were randomly assigned to receive either ACS treatment or normal saline (NS) dressings. The treatment was applied once a week for three weeks with a final assessment at three weeks from the first ACS application. A total of 30 patients took part in the study. Analysis of wound assessment data demonstrated statistically significant differences for wound surface area and Pressure Ulcer Scale for Healing scores (area score, exudate and tissue) from baseline to the end of the study in patients who received the ACS dressing, but not in patients who received the normal saline dressing. There were statistically significant differences in changes in: the wound surface area at week three (-6.4±2.69cm2 versus +0.4±2.52cm2); area score at week three (-2.2±1.08 versus +0.2±0.86); exudate at week two (-1.2±0.70 versus +0.0±0.45) and at week 3 (-1.3±0.72 versus -0.1±0.63); tissue at week two (-1.1±0.35 versus +0.0±0.53) and at week three (-1.8±0.65 versus -0.1±0.63); and the PUSH total score at week one (-1.6±0.98 versus +0.4±1.22), week two (-3.2±0.86 versus +0.4±0.98) and week three (-5.3±1.17 versus -0.0±1.33) between the ACS and NS groups, respectively. This trial revealed a significant decrease in wound surface area as well as a considerable improvement in wound healing in the ACS dressing group.

Evaluation of the autologous conditioned serum in the treatment of osteoarthritis

Aim: The present study was a retrospective study aiming to determine the effect of the autologous conditioned serum (ACS) on osteoarthritis (OA); we made this analysis by injecting it to a symmetrically involved knee.Methods: The present study comprised 33 patients (19 females, 14 males) with 66 knees and a mean age of 57.6±8.21 (range: 41-70). The patients included in the study had radiologically verified bilateral grade 2-3 OA of the knee according to Kellgren-Lawrence classification. Secondary arthritis, inflammatory joint diseases, clinically relevant hematologic or abnormal clinical chemistry values, joint instability, intra-articular corticosteroid injection within the previous 6 months, history of diabetes mellitus and body mass index greater than 30 kg/m2 were the exclusion criteria. Patients who had VAS difference more than 2 points between their knees were excluded from the study. ACS was injected twice a week for a total of 6 times in both knee joints for 3 weeks. The patients were analyzed with the Visual Analog Scale (VAS) (no pain was graded 0 and maximal pain was graded 10), the Knee Injury and Osteoarthritis Score (KOOS) (scoring ranges between 0 and 100. 0 indicates abnormally high level of knee problems, while 100 indicates a healthy knee with no problems) and the Knee Society Score (KSS) (Of the maximum 100 points, a possible 50 points are assigned to pain, 25 points to stability, and 25 points for range of motion) before the administration of the first injection and again 1 year after the last injection. Results: The pre-treatment and 1-year follow-up VAS values ​​of the patients were 7.36±0.93 (range: 5-9) and 3.27±1.23 (range: 1-6), respectively. ACS treatment showed a statistically significant decrease in VAS score (p &amp;lt;0.01). Pre-treatment and 1-year follow-up KOOS values ​​of the patients were 42.39±13.38 (range: 21-65) and 72.36±8.81 (range: 54-92), respectively. There was a statistically significant increase in the KOOS values of the patients (p &amp;lt;0.01). The pre-treatment and 1-year follow-up KSS values ​​of the patients were 42.79±10.26 (range: 14-61) and 70.61±9.32 (range: 49-84), respectively. There was a statistically significant increase in the KSS values of the patients (p &amp;lt;0.01). Conclusion: Intra-articular injection of ACS in patients with painful OA leads to significant improvements in pain severity, KOOS, KSS and DCS scores. In the light of these findings, ACS treatment may be considered as an effective and safe alternative treatment method in osteoarthritis.

Investigation of the biomechanical and histopathological effects of autologous conditioned serum on healing of Achilles tendon

ObjectiveThe aim of this study to evaluate the effects of autologous conditioned serum (ACS) on the healing of transected rat Achilles tendons via the assessment of biomechanical and histological parameters. MethodsThe study was conducted on 45 male Sprague–Dawley rats. Five rats were used as donors for ACS preparation. Animals were randomly assigned to the experimental or control group. In both groups, the Achilles tendon was cut transversally and then sutured. In the placebo control and ACS-treated groups, saline or ACS, respectively, was injected into the repair zone three times after surgery. Ten rats from each group (ACS group, n = 20; control group, n = 20) were euthanized at days 15 and 30 after surgery for histopathological (n = 5) and biomechanical (n = 5) testing. The histopathological findings were interpreted using the Bonar and Movin scales. Tendon remodelling was evaluated via the immunohistochemical staining of collagen type 3. Biomechanical effects were assessed by tensile testing. ResultsThe Bonar and Movin scale scores were significantly better in the ACS-treated group on both day 15 (p = 0.003 and p = 0.003, respectively) and day 30 (p = 0.005 and p = 0.004, respectively). The immunohistochemical density of collagen type 3 was significantly lower in the ACS-treated group on day 30 (p = 0.018). The type 1/3 collagen ratios of the groups were similar on days 15 and 30, as determined by Sirius Red staining (p = 0.910 and p = 0.133, respectively). In the biomechanical assessment results, the ACS-treated group's maximum load to failure values were significantly higher on day 15 (p = 0.049). ConclusionInjection of ACS had a positive effect on the histopathological healing of rat Achilles tendons on days 15 and 30 and on biomechanical healing on day 15. ACS treatment contributed to lowering the collagen type 3 density by day 30. According to our study, ACS may be favourable for the treatment of human Achilles tendon injuries and tendinopathies.

Comparison of clinical efficacy of platelet-rich plasma and autologous conditioned serum treatment in patients with osteoarthritis of the knee

Comparison of clinical efficacy of platelet-rich plasma and autologous conditioned serum treatment in patients with osteoarthritis of the knee

Evaluation of Two Protocols Using Autologous Conditioned Serum for Intra-articular Therapy of Equine Osteoarthritis—A Pilot Study Monitoring Cytokines and Cartilage-Specific Biomarkers

We hypothesised that shorter treatment intervals of intraarticular autologous conditioned serum (ACS) injections would more beneficially affect the synovial fluid (SF) concentrations of IL-1ra, IL-1β and cartilage biomarkers, compared with the traditional weekly treatment intervals in joints suffering from natural OA. In a randomised comparative study, 12 horses with OA were allocated to two groups (n = 6). The horses in group 1 received three intraarticular ACS injections at weekly intervals, whereas the horses in group 2 received three intraarticular ACS injections at two-day intervals. The levels of IL-1ra, IL-1β, CPII, C12C and CS 846 were determined in SF before and after ACS treatment using commercially available ELISA kits. The SF IL-1ra concentration 1 hour and 4 hours after ACS injection was significantly increased compared to baseline levels and decreased back to it within 48 hours. Comparing the SF IL-1ra, IL-1β, C12C, CS 846 and CP II levels before and 42 days after ACS treatment, group 2 showed a significant decrease in all parameters and an approximation on the levels in normal joints. These results indicate that the long-time effect of an ACS treatment given at two-day intervals is characterized by decreased SF IL1ra, IL-1β, C12C, CP II and CS 846 concentrations, which might indicate an improvement in joint inflammation and cartilage degrading processes . Further investigations with greater sample sizes have to prove if the two-day treatment interval is preferable to the widely used treatment protocol of weekly intraarticular ACS injections.

Elevated CRP level could herald less efficient autologous conditioned serum (ACS) treatment

Autologous conditioned serum (ACS) is a biologically based local treatment aiming to influence the cytokine imbalance and is used in a variety of orthopedic diseases and conditions. The ACS contains elevated levels of various anti-inflammatory cytokines, such as IL-1 RA (receptor antagonist), IL-4 and IL-10 and several growth factors. It contains a combination of cytokines and growth factors, and their specific contribution to clinical effects have yet to be determined. Serum conditioned in that specific way does not always have the same content and concentration of the anti-inflammatory cytokines and growth factors. We hypothesize that ACS should not be prepared and administered if elevated C-reactive protein (CRP) levels are present at the moment of obtaining the patient’s blood because of the potential detrimental effect of elevated pro-inflammatory cytokines in the same blood, namely IL-1 and TNF. We propose introduction of CRP measuring before any ACS treatment. The cut off value would be set at 5mg/dL as an usual value suggesting inflammation. Avoidance of collecting and administering ACS if elevated CRP is present would potentially eliminate low quality ACS.

Effect of a single injection of autologous conditioned serum (ACS) on tendon healing in equine naturally occurring tendinopathies.

IntroductionAutologous blood-derived biologicals, including autologous conditioned serum (ACS), are frequently used to treat tendinopathies in horses despite limited evidence for their efficacy. The purpose of this study was to describe the effect of a single intralesional injection of ACS in naturally occurring tendinopathies of the equine superficial digital flexor tendon (SDFT) on clinical, ultrasonographic, and histological parameters.MethodsFifteen horses with 17 naturally occurring tendinopathies of forelimb SDFTs were examined clinically and ultrasonographically (day 0). Injured tendons were randomly assigned to the ACS-treated group (n = 10) receiving a single intralesional ACS injection or included as controls (n = 7) which were either untreated or injected with saline on day 1. All horses participated in a gradually increasing exercise programme and were re-examined nine times at regular intervals until day 190. Needle biopsies were taken from the SDFTs on days 0, 36 and 190 and examined histologically and for the expression of collagen types I and III by immunohistochemistry.ResultsIn ACS-treated limbs lameness decreased significantly until day 10 after treatment. Swelling (scores) of the SDFT region decreased within the ACS group between 50 and 78 days after treatment. Ultrasonographically, the percentage of the lesion in the tendon was significantly lower and the echogenicity of the lesion (total echo score) was significantly higher 78 and 106 days after intralesional ACS injection compared to controls. Histology revealed that, compared to controls, tenocyte nuclei were more spindle-shaped 36 days after ACS injection. Immunohistochemistry showed that collagen type I expression significantly increased between days 36 and 190 after ACS injection.ConclusionsSingle intralesional ACS injection of equine SDFTs with clinical signs of acute tendinopathy contributes to an early significant reduction of lameness and leads to temporary improvement of ultrasonographic parameters of repair tissue. Intralesional ACS treatment might decrease proliferation of tenocytes 5 weeks after treatment and increase their differentiation as demonstrated by elevated collagen type I expression in the remodelling phase. Potential enhancement of these effects by repeated injections should be tested in future controlled clinical investigations.

Autologous IL1-Ra and several growth factors influence on result after double bundle ACL surgery

Bone tunnel widening (BTW) appear after knee anterior cruciate ligament (ACL) single bundle technique (SB) reconstruction and could result in ligament laxity or lead to increased failure rates. It could be affected on biomechanical and biomolecular base. Our aim was to establish whether double bundle technique (DB) in combination with biologic treatment of intra-articular application of autologous conditioned serum (ACS) containing endogenous anti-inflammatory cytokines including IL-1Ra and several growth factors, could enhance result of ACL surgery. In prospective, randomized, double-blind trial with four parallel groups, 124 patients were treated. We compared tibial BTW measured by CT-scans in three different postoperative periods. The clinical efficacy was assessed by patient administered outcome instruments (IKDC 2000, LYSHOLM, TEGNER) up to two years following ACL reconstruction in 4 groups of patients regarding different combination of treatment: – A: SB + Placebo; – B: SB + ACS; – C: DB + Placebo; – D: DB + ACS. Postoperative tibial tunnel diameters in all groups were significantly larger than they were directly after surgery. BTW was significantly less in group D than in other groups (P < 0.005). Clinical outcomes were consistently better in patients treated with DB technique, especially in group D, at all data points and for all outcome parameters. Combination of biologic ACS treatment and DB ACL reconstruction give the best postoperative knee stability and influence function recovery and clinical result by affecting BTW. The cause and prevention of BTW must be understood by surgeon to improve surgical technique, choice of graft methods and postoperative treatment regimens.

Osteoarthritis treatment using autologous conditioned serum after placebo

Background and purpose — Autologous conditioned serum (ACS) is a disease-modifying drug for treatment of knee osteoarthritis, and modest superiority over placebo was reported in an earlier randomized controlled trial (RCT). We hypothesized that when given the opportunity, placebo-treated patients from that RCT would now opt for ACS treatment, which would result in a greater clinical improvement than placebo. Methods — Of 74 patients treated with placebo in the previous trial, 20 opted for ACS treatment. Patients who did not choose further treatment were interviewed about their reasons. Clinical improvement of the 20 ACS-treated patients was measured using knee-specific clinical scores, as was “response shift” at 3 and 12 months. Results — In the 20 patients who did opt for ACS, the visual analog scale (VAS) score for pain improved; but after 12 months, clinical results were similar to those after placebo treatment. Response shift measurement demonstrated that the 20 patients had adapted to their disabilities during treatment. Interpretation — Placebo-treated patients from an earlier trial were reluctant to undergo ACS treatment, in part due to the laborious nature of the therapy. In a subset of patients who opted for treatment, ACS treatment after placebo did not result in greater clinical improvement than placebo treatment only. However, due to the limited power of the current study and possible selection bias, definite advice on using or refraining from ACS cannot be given.

AB0981 Comparison of the efficacy of autologous conditioned serum and hyaluronic acid treatment in hip osteoarthritis

BackgroundIn the synovial fluid of joints affected by osteoarthritis (OA),the concentration and molecular weight of hyaluronic acid (HA) is reduced,which negatively affects its viscoelastic properties and lubricity.In this regard,the possibility...

Effects of serum and autologous conditioned serum on equine articular chondrocytes treated with interleukin-1 β

To compare the effects of autologous equine serum (AES) and autologous conditioned serum (ACS) on equine articular chondrocyte metabolism when stimulated with recombinant human (rh) interleukin (IL)-1β. Articular cartilage and nonconditioned and conditioned serum from 6 young adult horses. Cartilage samples were digested, and chondrocytes were isolated and formed into pellets. Chondrocyte pellets were treated with each of the following: 10% AES, 10% AES and rhIL-1β, 20% AES and rhIL-1β, 10% ACS and rhIL-1β, and 20% ACS and rhIL-1β, and various effects of these treatments were measured. Recombinant human IL-1β treatment led to a decrease in chondrocyte glycosaminoglycan synthesis and collagen II mRNA expression and an increase in medium matrix metalloproteinase-3 activity and cyclooxygenase-2 mRNA expression. When results of ACS and rhIL-1β treatment were compared with those of AES and rhIL-1β treatment, no difference was evident in glycosaminoglycan release, total glycosaminoglycan concentration, total DNA content, or matrix metalloproteinase-3 activity. A significant increase was found in chondrocyte glycosaminoglycan synthesis with 20% AES and rhIL-1β versus 10% ACS and rhIL-1β. The medium from ACS and rhIL-1β treatment had a higher concentration of IL-1β receptor antagonist, compared with medium from AES and rhIL-1β treatment. Treatment with 20% ACS and rhIL-1β resulted in a higher medium insulin-like growth factor-I concentration than did treatment with 10% AES and rhIL-1β. No difference in mRNA expression was found between ACS and rhIL-1β treatment and AES and rhIL-1β treatment. Minimal beneficial effects of ACS treatment on proteoglycan matrix metabolism in equine chonrocytes were evident, compared with the effects of AES treatment.

Gambling on putative biomarkers of osteoarthritis and osteochondrosis by equine synovial fluid proteomics

Osteoarthritis (OA) and osteochondrosis (OC) are two of the main challenges in orthopedics, whose definitive diagnosis is usually based on radiographic/arthroscopic evidences. Their early diagnosis should allow preventive or timely therapeutic actions, which are generally precluded from the poor relationships occurring between symptomatologic and radiographic evidences. These limitations should be overcome by improving the knowledge on articular tissue metabolism and on molecular factors regulating its normal homeostasis, also identifying novel OA and OC biomarkers suitable for their earlier diagnoses, whenever clinical/pathological inflammatory scenarios between these joint diseases seem somewhat related. To identify proteins involved in their aetiology and progression, we undertook a differential proteomic analysis of equine synovial fluid (SF), which compared the protein pattern of OA or OC patients with that of healthy individuals. Deregulated proteins in OA and OC included components related to inflammatory state, coagulation pathways, oxidative stress and matrix damage, which were suggestive of pathological alterations in articular homeostasis, plasma-SF exchange, joint nutritional status and vessel permeability. Some proteins seemed commonly deregulated in both pathologies indicating that, regardless of the stimulus, common pathways are affected and/or the animal joint uses the same molecular mechanisms to restore its homeostasis. On the other hand, the increased number of deregulated proteins observed in OA with respect to OC, together with their nature, confirmed the high inflammatory character of this disease. Some deregulated proteins in OA found a verification by analyzing the SF of injured arthritic joints following autologous conditioned serum treatment, an emergent therapy that provides positive results for both human and equine OA. Being the horse involved in occupational/sporting activities and considered as an excellent animal model for human joint diseases, our data provide suggestive information for tentative biomedical extrapolations, allowing to overcome the limitations in joint size and workload that are typical of other small animal models.

Effect of BMP-12, TGF-β1 and autologous conditioned serum on growth factor expression in Achilles tendon healing

Achilles tendon ruptures are devastating and recover slowly and incompletely. There is a great demand for biomolecular therapies to improve recovery, yet little is understood about growth factors in a healing tendon. Here, the role of growth factors during tendon healing in a rat model and their reaction to single and multiple growth factor treatment are explored. Rat tendons were transected surgically and resutured. The expression of bFGF, BMP-12, VEGF and TGF-β1 was assessed by immunohistochemical analysis one to 8 weeks after surgery. Paracrine effects of TGF-β1 or BMP-12 added by adenoviral transfer, as well as the effect of autologous conditioned serum (ACS) on growth factor expression, were evaluated. bFGF, BMP-12 and VEGF expression was highest 1 week after transection. bFGF and BMP-12 declined during the remaining period whereas VEGF expression persisted. TGF-β1 expression dramatically increased after 8 weeks. ACS treatment increased bFGF (P = 0.007) and BMP-12 (P = 0.004) expression significantly after 8 weeks. Also overall expression of bFGF, BMP-12 and TGF-β1 regardless of time point was significantly greater than controls with ACS treatment (P < 0.05). Both BMP-12 and TGF-β1 treatments had no significant effect. No effect was observed in VEGF with any treatment. bFGF, BMP-12, VEGF and TGF-β1 are differentially expressed during tendon healing. Additional BMP-12 or TGF-β1 has no significant influence, whereas ACS generally increases expression of all factors except VEGF. Staged application of multiple growth factors may be the most promising biomolecular treatment.

Response to: cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra-articular levels after injection - authors' reply

We thank Moser for his comments [1] on the paper [2] we published in a previous issue of Arthritis Research & Th erapy. To dispel any concerns about the proper use of the product, the autologous conditioned serum (ACS) in this study was prepared in a GMP (good manufacturing practice) facility in strict accordance with the guidelines supplied by the manufacturer (Orthogen, Dusseldorf, Germany) and was injected six times at 3-day intervals in accordance with the instructions given. As recommended, immediately before injection of ACS, synovial fl uid was carefully aspirated to minimize ACS dilution. Th is synovial fl uid was used to determine the cytokine levels before and during ACS treatment. We showed that, 3 days after injection, cytokine levels were at baseline and had no secondary eff ects on other cyto kines. Th is fi nding is not in confl ict with that of Darabos and colleagues [3], whose publication is cited by Moser; upon careful reading of that publication, none of the eff ects of ACS injection, including the alleged decrease in synovial fl uid levels of interleukin-1 (IL-1), turned out to be statistically signifi cant. We certainly agree with Moser that in vitro and in vivo studies should be well controlled. However, in the case of ACS, the use of an autologous product is not required; this autologous product is devoid of cells, which are the only factors capable of evoking an immune response upon transfer of human materials to human recipients. We have used unconditioned serum as controls in our in vitro studies because serum in general seems to be more amenable to cartilage health than either saline or the synovial fl uid the tissue is exposed to in vivo [4]. In this setup, we could not demonstrate any eff ect of conditioning the serum. Th e observation that IL-1 and tumor necrosis factor-alpha levels were upregulated in ACS, in contrast to the levels found previously upon characterization of ACS, may be due to the use of healthy subjects in the latter case, whereas we characterized the ACS prepared from osteoarthritis (OA) patients, the intended target population. Blood from OA patients was recently

Response to: cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra-articular levels after injection

We welcome the interest of Rutgers and colleagues [1] in providing additional information on the mode of action of autologous conditioned serum (ACS). Studies such as the one they published in a recent issue of Arthritis Research & Th erapy are important because the Orthokine ACS treatment (Orthogen, Dusseldorf, Germany) has been shown to be safe and eff ective in a number of clinical studies and is used widely in Europe. Randomized controlled trials have confi rmed the effi cacy of this treatment in osteoarthritis of the knee and lumbar radiculopathy. Additional human studies show promise in treating muscle injury and tunnel widening after anterior cruciate ligament (ACL) reconstruction. All clinical and preclinical studies confi rm an excellent riskto-benefi t ratio. We would like to off er some comments concerning the paper of Rutgers and colleagues. Th e method of producing ACS has been carefully optimized to enrich for anti-infl ammatory cytokines, such as interleukin (IL)-1Ra, IL-10, and IL-13, while keeping low the concentration of infl ammatory cyto kines, such as IL-1β and tumor necrosis factor-alpha (TNF-α). Th e ACS preparation of Rutgers and colleagues had increased concen trations of IL-1β and TNF-α, and this raises questions about the composition of the product that was tested. Nevertheless, despite the elevated concentrations of these two cytokines, there was no adverse eff ect of ACS on proteoglycan turnover in the cartilage explant cultures. Th is suggests that anti-infl ammatory and possibly chondro protective ingredients within ACS pre dominate. Further studies using additional controls and autologous instead of heterologous serum would be interesting. Th e third component of the study by Rutgers and colleagues measured cytokine levels in synovial fl uids before and after treatment with ACS. No changes were found, but it should be noted that Orthokine should be used only when knee eff usion has been removed eff ectively. Th e described possible aspiration of synovial fl uid after treatment with ACS raises questions about the selection of these patients or the composition of the injected ACS. In this regard, it is worth noting that another study [2,3] involving the injection of Orthokine ACS into knees after ACL plasty showed evidence of reduced IL-1β content in synovial fl uid. Th is reduction corresponded with im proved pain and function and with reduced tunnel widening. Clearly, the mechanisms through which ACS brings about clinical improvement are incompletely understood and should be the subject of additional research.