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K. Dean Reeves, M.D. Clinical Associate Professor Physical Medicine and Rehabilitation
Emphasis on Research in Use of Prolotherapy (Also called Regenerative Injection Therapy) ReevesOffice @ gmail.com Office Email DeanReevesMD @ gmail.com Personal email
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Animal Studies
Dex: Jensen el al 2008 Jensen et al 2008 (2nd article) Dex: Park et al 2007 Dex: KIm SA 2006 Dex: Kim HJ 2003 Dex: Kim HJ 2006
ABSTRACT: Prolotherapy is an alternative injection-based therapy for chronic musculoskeletal pain. Three different proliferants, D-glucose (dextrose), phenol-glucose-glycerine (P2G), and sodium morrhuate, used in prolotherapy are hypothesized to strengthen and reorganize chronically injured soft tissue and decrease pain through modulation of the inflammatory process. Our hypothesis is that commonly used prolotherapy solutions will induce inflammation (leukocyte and macrophage infiltration) in medial collateral ligaments (MCLs) compared to needlestick, saline injection, and no-injection controls. MCLs of 84 Sprague- Dawley rats were injected one time at both the tibial and femoral insertions. Immunohistochemistry (IHC) was used to determine the inflammatory response at three locations (tibial and femoral insertions and midsubstance) 6, 24, and 72 h after dextrose injection compared to saline- and no-injection controls and collagenase (positive control) (n = 4). qPCR was used to analyze gene expression 24 h postinjection (n = 4). Sodium morrhuate, P2G, and needlestick control were also investigated after 24 h (n = 4). In general, inflammation (CD43+, ED1+, and ED2+ cells) increased after prolotherapy injection compared to no-injection control but did not increase consistently compared to saline and needlestick control injections. This response varied by both location and proliferant. Inflammation was observed at 6 and 24 h postinjection but was resolved by 72 h compared to no-injection controls (p < 0.05). CD43+ leukocytes and ED2+ macrophages increased compared to needlestick and saline-injection control, respectively, 24 h postinjection (p < 0.05). Prolotherapy injections created an inflammatory response, but this response was variable and overall, not uniformly different from that caused by saline injections or needlestick procedures. [© 2008 Orthopaedic Research Society.]. AUTHOR'S ADDRESS: Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin, USA. david.rabago@fammed.wisc.edu. Home Physicians Animal Research
This study was limited by a small size due to expense and was limited in the number of time intervals at which the rats could be studied. The demonstration of increased ligament size needs to be further explored and the scientific integrity of this study is to be applauded. Although healing in rats is faster than humans, expecting mechanical changes by 2 weeks may not be reasonable. Note that the only long term human followup study of machine measured ligament laxity demonstrated a 54% improvement in KT1000 values for ACL laxity (side to side anterior displacement comparison) by 1 year and 72% by 3 years in patients with ACL laxity. Reeves KD, Hassanein K. Long term effects of dextrose prolotherapy for anterior cruciate ligament laxity: A prospective and consecutive patient study. Altern Ther Health Med (United States), May-Jun 2003, 9(3) p58-62. I look forward to further study as funds allow. Again, here is the abstract Jensen KT; Rabago DP; Best TM; Patterson JJ; Vanderby R. Response of knee ligaments to prolotherapy in a rat injury model.: Am J Sports Med (United States), Jul 2008, 36(7) p1347-57 AUTHOR'S ADDRESS: Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin, USA. ABSTRACT: BACKGROUND: Prolotherapy is an alternative therapy for chronic musculoskeletal injury including joint laxity. The commonly used injectant, D-glucose (dextrose), is hypothesized to improve ligament mechanics and decrease pain through an inflammatory mechanism. No study has investigated the mechanical effects of prolotherapy on stretch-injured ligaments. HYPOTHESES: Dextrose injections will enlarge cross-sectional area, decrease laxity, strengthen, and stiffen stretch-injured medial collateral ligaments (MCLs) compared with controls. Dextrose prolotherapy will increase collagen fibril diameter and density of stretch-injured MCLs. STUDY DESIGN: Controlled laboratory study. METHODS: Twenty-four rats were bilaterally MCL stretch-injured, and the induced laxity was measured. After 2 weeks, 32 MCLs were injected twice, 1 week apart, with either dextrose or saline control; 16 MCLs received no injection. Seven uninjured rats (14 MCLs) were additional controls. Two weeks after the second injection, ligament laxity, mechanical properties (n = 8), and collagen fibril diameter and density (n = 3) were assessed. RESULTS: The injury model created consistent ligament laxity (P < .05) that was not altered by dextrose injections. Cross-sectional area of dextrose-injected MCLs was increased 30% and 90% compared with saline and uninjured controls, respectively (P < .05). Collagen fibril diameter and density were decreased in injured ligaments compared with uninjured controls (P < .05), but collagen fibril characteristics were not different between injured groups. CONCLUSION: Dextrose injections increased the cross-sectional area of MCLs compared with saline-injected and uninjured controls. Dextrose injections did not alter other measured properties in this model. CLINICAL RELEVANCE: Our results suggest that clinical improvement from prolotherapy may not result from direct effects on ligament biomechanics.Home Dex: Park et al 2007 10%
Dextrose injection protects cartilage in
rabbits after cutting
the AC Park Y, Lim S, Lee I, Lee T, Kim T, Han JS. Intra-articular injection of a nutritive mixture solution protects articular cartilage from osteoarthritic progression induced by anterior cruciate ligament transection in mature rabbits: a randomized controlled trial. Arthritis Research & Therapy 2007. 9(1):R8 This study involved 24 mature New Zealand
Rabbits. The RIGHT knee of each rabbit was considered a normal control and the
LEFT knee was altered by cutting the ACL ligament which leads to arthritic
changes in 3-8 weeks in these animals. Rabbits were
randomized to either Dextrose 10% injection or Saline injection (0.5 ml)
at 6 weeks, 8, 10, 13 and 16 weeks. Animals were sacrificed
at 19 weeks and cartilage samples were then randomly taken from the right knees
with intact ACL ligament, left knees injected with dextrose and left knees
injected with saline. H&E staining and SEM (Scanning
electron microscopy) were performed. Here is the representative H&E
result. 2A: Knee with normal ACL ligament. Note no significant surface irregularity. 2B: Knee with cut ACL, injected with dextrose. Moderate surface irregularity is seen with some swelling of cartilage cells and extra cells. 2C: Knee with cut ACL, injected with saline. Severe surface irregularity is seen with cartilage cell loss and loss of cartilage down to bone. The Mankin grading method for osteoarthritis was performed and no significant differences in cartilage erosion were seen in control versus dextrose injection rabbits with cut ACL, but significant differences were seen in rabbit knees with cut ACL and saline injection.Two special notes. Although the authors included amino acids with the injection there is not evidence from other literature that these have a separate effect; these appear merely to be included for proprietary reasons. Abstract (see PDF in folders) Osteoarthritis (OA) is a degenerative disease which disrupts the collagenous matrix of articular cartilage, and is difficult to cure because articular cartilage is a nonvascular tissue. Treatment of OA has targeted macromolecular substitutes for cartilage components, such as hyaluronic acid or genetically engineered materials. However, the goal of this study is to examine whether intra-articular injection of the elementary nutrients restores the matrix of arthritic knee joints of mature animals. A nutritive mixture solution (NMS) was composed of elementary nutrients such as glucose or dextrose, amino acids and ascorbic acid. It was administered five times, at the 6th, 8th, 10th, 13th, and 16th weeks, into the unilateral anterior cruciate ligament transected (ACLT) knee joints of mature New Zealand White rabbits. It was compared to normal saline (NS)-injection effect. OA progression was histopathologically evaluated by hematoxylin & eosin (H&E) staining, by the Mankin grading method, and by scanning electron microscopy (SEM) at the 19th week. NMS-injection decreased progressive erosion of articular cartilage overall compared to NS-injection (p<0.01), and showed no differences compared to normal cartilage which did not undergo ACLT, by Mankin grading method. H&E staining and SEM results also showed that NMS-injection, as apposed to NS-injection, restored the cartilage matrix that is known to be composed of a collagen and proteoglycan (PG) network. Thus, NMS-injection is a potent treatment that significantly retards OA progression, which in turn prevents progressive destruction of joints and functional loss in mature animals. Home Physicians Animal Research
Abstract: In this pilot study, hypertonic dextrose solution was used to induce fibrosis of the subsynovial connective tissue (SSCT) and create an animal model of potential use in the study of carpal tunnel syndrome (CTS). The SSCT of the carpal tunnel in 15 New Zealand white rabbits were injected with 0.05 ml of 10% dextrose solution in 1 paw and 0.05 ml of saline in the contralateral paw, to serve as a control. The animals were killed at 1, 2, 4, 8, or 12 weeks. While the saline side showed minimal changes at any time period, the hypertonic dextrose side showed progressive noninflammatory SSCT fibrosis, with vascular proliferation and thickening of collagen bundles. Demyelination of the median nerve developed at 12 weeks after the injection on the dextrose side. These findings are similar to the progression of pathology noted in humans with CTS. Home Physicians Animal Research Dex: Kim HJ 2006 Injection
of 20% dextrose in normal Achilles tendon led to an increase in fibril diameter
and fibroblast proliferation in rats, and this was not stopped by oral NSAIDs.
This indicates that injection with dextrose causes proliferation via mechanisms which are
not merely inflammatory. Kim HJ, Kim SH, Yun DH, Lee KS, Jeong TS. The Effects of Anti-inflammatory Drugs on Histologic Findings of the Experimental Prolotherapy Model. J Korean Acad Rehab Med. 2006 Aug:30(4):378-384 From rehab medicine Departments in Eulji Univ School of Medicine and the Department of Pathology at Yonsei Univeristy College of Medicine. This study demonstrated proliferation effects (increase in transverse diameter and count of fibroblasts in Achilles tendon of rats injected 3 times at 1 week interval with 20% dextrose and rats sacrificed at 3 and 6 week after injection 1. Opposite non injected Achilles was the control. Three groups (Tylenol, NSAIDS and no med group) all turned out the same. This further reinforces proliferant effects of dextrose and potentially injection itself which are not via inflammatory mechanisms. Here is the abstract. Kim HJ, Kim SH, Yun DH, Lee KS, Jeong TS. The Effects of Anti-inflammatory Drugs on Histologic Findings of the Experimental Prolotherapy Model. J Korean Acad Rehab Med. 2006 Aug:30(4):378-384 Department of Rehabilitation Medicine, Eulji
Hospital, Eulji University School of Medicine, Korea. cynikid@naver.com Home Physicians Animal Research Dex: Kim SA 2006
Both 10% dextrose and Autologous serum lead
to tissue regeneration in Kim SA, Kim EH, Kim SY, Lee SY, Yoon JN, Lee YK. The Effects of Hyperosmolar Dextrose and Autologous Serum Injection in the Experimental Articular Defect of Rabbit. J. Korean Acad Rehabil Med 2006 Apr;30(2):173-178. Korean From the Department of Rehabilitation Medicine, Soonchunhyang University College of Medicine, Korea. sooapmr@schch.co.kr and the Department of Pathology, Soonchunhyang University College of Medicine, Korea. In classic fashion, lesions were created in articular cartilage in rabbit and then no treatment or dextrose 10% or autologous serum were administered with evidence of repair in both the dextrose and autologous serum treated groups, but none in the no treatment group. I have communicated with the authors by email with the following questions: 1. What were the width and depth of cartilage lesions you induced and where were the defects placed? 2. The nature of the cartilage tissue that was translucent in the dextrose and serum groups? 3. Were the RBCs spun down and removed so that you were using platelet poor plasma for the plasma group? 4. What was the treatment method of these animals. IE Injection how many times prior to 6 week findings? 5. Was it 6 weeks after the last treatment that the defects were observed? 6. Were there any other changes on the surface of the denuded cartilage? Request for information was on Nov 19, 2007. Here is the abstract. Kim SA, Kim EH, Kim SY, Lee SY, Yoon JN, Lee YK. The Effects of Hyperosmolar Dextrose and Autologous Serum Injection in the Experimental Articular Defect of Rabbit. J. Korean Acad Rehabil Med 2006 Apr;30(2):173-178. Korean OBJECTIVE: Although the clinical effects of prolotherapy on osteoarthritis has been reported, there have been few previous studies showing the effects as a proliferant on articular cartilage. Also the autologous blood has been reported to used as a growth factor stimulant recently, we were trying to use dextrose and autologous serum for tissue regeneration respectively and evaluated the proliferative effect of autologous serum comparing with that of dextrose. METHOD: Twenty four rabbits were used for this study. The rabbits were divided into three groups. Group A did not get any special treatment. Group B was treated with 10% dextrose and group C with autologous serum. Six weeks later, gross appearance and histologic findings were evaluated. RESULTS: After sacrifice, the gross inspection of the knee joints revealed that groups B and C were filled with the translucent tissue in defective cartilage. Group A still had defective cartilage. Histologic evaluation revealed increase of cellularity in the defect of the injected specimens when compared with the control. There was no morphological difference between group B and C. CONCLUSION: The repair process of the articular cartilage defects using dextrose and autologous serum were shown to be more effective than that of control group. Home Physicians Animal Research Dex: Kim HJ 2003
Dextrose 5% and 20% cause proliferation in rat
Achilles tendon Kim HJ, Jeong TS, Kim WS, Park YS. Comparison of Histological Changes in Accordance with the Level of Dextrose-Concentration in Experimental Prolotherapy Model. J Korean Acad Rehabil Med. 2003 Dec;27(6):935-940. Korean. Kim et al in 2003 published a work on animal Achilles in which injection of identical osmolarity solutions (1,110mOSM) were injected around the right Achilles tendon of rats. Group A 20% dextrose Group B 5% Dextrose in NaCL Group C NaCL At six weeks animals were sacrificed and both transverse diameter of fibroblasts and count of fibroblasts was measured in blind fashion. Significant differences between controls were only in Dextrose 5 and 20%, and these two groups did not show significant differences between each other. Questions from me were answered by Tae-Seok Jeong, M.D, M.M.Sc, who is currently in Liverpool, and available at tessj@hanmail.net . The responses indicated that each rat was injected only on one occasion. The injections were about the right Achilles tendon at four different sites. These included tendon sheath from both sides, musculotendinous junction and tendinosseous junction. Left Achilles was not injected and served as the control. They did not attempt to inject tendon directly although for tendinoosseous junction the calcaneal bone was touched and then withdrawn slightly for injection. Groups A and B had significant differences between injected and non injected sides, but in group C there were not significant differences between control and injected sites. No intergroup differences were noted. (By Kruskall-Wallis test) Specimens were only of tendon. Further questions can be answered via Dr. Kim, Hyun Jung at khj2603@eulji.ac.kr, or hyunnykim@hanmail.net. Here is the abstract: From the Department of Rehabilitation Medicine, Eulji University School of Medicine, Korea. tessj@hanmail.net Department of Pathology, Eulji University School of Medicine, Korea Kim HJ, Jeong TS, Kim WS, Park YS. Comparison of Histological Changes in Accordance with the Level of Dextrose-Concentration in Experimental Prolotherapy Model. J Korean Acad Rehabil Med. 2003 Dec;27(6):935-940. Korean. OBJECTIVE: Comparing histological changes according to the level of dextrose-concentration of proliferant under the same osmolarity on Achilles tendon of rat. METHOD: One millimeter of three proliferant solutions (20% dextrose water-group A, 5% dextrose water mixed with NaCl-group B, NaCl solution-group C) with the same osmolarity (1,110 mOsm) was injected around the right Achilles tendon of each rat, whereas the left was not injected to be used as control. After six weeks of injection, the injected tendons and controls were obtained. The transverse diameter of gross specimen, the count of fibroblasts on light microscope, and the findings of cross- sectional analysis using electron microscope were compared. RESULTS: Overall, transverse diameter and the count of fibroblasts increased in the injected specimens compared to controls, however, their significant differences were demonstrated only for the two groups injected with dextrose containing solutions (p<0.05). However, A and B groups did not show significant differences in all parameters investigated. On electron micrograph, fibril diameters of solution- injected tendon consisted of either extremely large or small sizes with the limited intermediate sizes. CONCLUSION: Although high osmolar solution could increase the transverse diameter and fibroblast counts, however, dextrose-containing solution was much more effective as a proliferant solution. Home Physicians Animal Research
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DEX. Jenson et al 2008.
Inflammation from any needle insertion with or without injection. Needle
insertion without injection, injection saline and injection of dextrose all
produced an inflammatory reaction in rat knee ligaments. This means that saline injection
and injection with a needle are not a placebo. This is very
important in design of clinical studies.
Regardless of what caused the inflammation, the inflammation
lasted only 24 hours. Note however that animals go through healing
phases faster than humans.
Jensen KT, Rabago DP, Best TM, Patterson JJ, Vanderby R Early inflammatory
response of knee ligaments to prolotherapy in a rat model. J Orthop Res, Jun
2008, 26(6) p816-23 
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