Animal Research

Animal Research

 

  Bone Marrow Aspirate Concentrate injection appears to help repair full thickness knee cartilage defects after microfracture. A small pilot study in horses.

Concentrated bone marrow aspirate improves full-thickness cartilage repair. Fortier L, Potter H, Rickey E, Schnabel L, Ellsworth J, Foo L, Nixon A. OSA meeting 2009.

Dr. Reeves' Notes:  Ten mixed breed, young adult horses (2-5 years of age; approx. 450kg) received a full thickness cartilage defect in the lateral trochlear region of each femur and only one side was injected with bone marrow aspirate concentrate (BMAC) Re-check arthroscopy was performed 16 weeks after surgery. Horses were euthanized 8 months from the initial surgery. Imaging: MRI of the knee joints was conducted on a 3 Tesla clinical system. Subsequent to MRI, the joints were opened and photographed. Synovial membrane and osteochondral sections were obtained for histology including H&E, toluidine blue, safranin-O and fast green, and type II collagen immunochemistry. Sections were scored using the ICRS scoring system. The MRIs were assessed for percentage fill, synovial reaction and subchondral bony reaction. No horses developed inflammation or infection in any joint. Recheck arthroscopy showed significantly improved repair tissue in the BMAC grafted site compared to microfracture (mean±sem: BMAC 8.8±1.2, control 16.2±0.49; p=0.0004). The repair tissue remained improved in the BMAC grafted site at necropsy 8 months following surgery (mean total histology score±sem: BMAC 7.1±1.2, control 14.1±1.1 p=0.0006). MRI evaluation indicated significantly increased GAG and collagen content (Table 1), and improved fill in BMAC grafted defects. Histological evaluation supported MRI evaluation of improved percent fill, GAG and collagen content in BMAC grafted defects compared to controls (Figure 2, Table 2). There was also agreement that there was no synovial reaction due to BMAC grafting.

The study suggests that BMAC provides a method to improve cartilage repair with respect to filling of the defect as well as GAG and type II collagen content. Further, there are no apparent adverse reactions to BMAC grafting which is durable for at least 8 months after surgery. Further studies are required to determine the durability of BMAC grafts after exercise and in humans.

 

Early inflammatory response of knee ligaments to prolotherapy in a rat model.

Jensen KT, Rabago DP, Best TM, Patterson JJ, Vanderby R  J Orthop Res, Jun 2008, 26(6) p816-23

 

Dr. Reeves' Notes:  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.

The abstract is available below...

SUMMARY: 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

 

Response of knee ligaments to prolotherapy in a rat injury model (2008)

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

Dr. Reeves' Notes:  A model of MCL injury was used. Two weeks and three weeks after injury rats were injected in the injured area with either saline or dextrose. Two weeks after that the animals were sacrificed. No differences in collagen size between groups was seen, although the dextrose injected ligaments were 30% bigger than the saline groups and 70% bigger than uninjured controls. Ligament laxity was still present but followup time was limited (only 2 weeks after the last injection).

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.

The abstract is available below...

AUTHOR'S ADDRESS: Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin, USA.

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. 

 

 10% Dextrose injection protects cartilage in rabbits after cutting the ACL

 

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

Dr. Reeves' Notes: 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.

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.

 

 

 Prolotherapy & Carpal Tunnel Syndrome: A Rabbit Study

Oh S, Ettema AM, Zhao C, Zobitz ME, Wold LE, An Kai-Nan, Amadio PC. Dextrose-Induced Subsynovial Connective Tissue Fibrosis in the Rabbit Carpal Tunnel: A Potential Model to Study Carpal Tunnel Syndrome. Hand 3(1):2008 Pgs 34-40.

Dr. Reeves' Notes: Proof of proliferation of new tendon/ligament (collagen) tissue in rabbit. Injection of 10% dextrose just once in the rabbit carpal tunnel equivalent causes growth of new collagen tissue, thickening the roof over the carpal tunnel.

The abstract is available below...

Summary: 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.

 

 

 

 

Prolotherapy & Achilles Tendon: A Rat Study

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

Dr. Reeves' Notes:   From rehab medicine Departments in Eulji Univ School of Medicine and the Department of Pathology at Yonsei University 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.

Department of Rehabilitation Medicine, Eulji Hospital, Eulji University School of Medicine, Korea. cynikid@naver.com
Department of Pathology, Yonsei University College of Medicine, Korea.
Department of Physical Medicine & Rehabilitation Medicine, Andong General Hospital, Korea.

The abstract is available below...

OBJECTIVE: To investigate the effects of nonsteroidal anti- inflammatory drugs (NSAIDs) and acetaminophen on histologic changes of the Achilles tendon in an experimental prolotherapy model.

METHOD: The right Achilles tendon of 60 rats was injected with 20% dextrose on experimental day 1, 7, and day 14, whereas the left was not injected and used as control. Rats were divided into 3 subgroups: NSAIDs medication group (10 mg/kg/day), acetaminophen medication group (100 mg/ kg/day) and no medication group. Medications were given for 3 consecutive days after each injection. Rats were sacrificed at 3 and 6 weeks after first injection. The transverse diameter of the gross specimens, the number of fibroblasts on light microscope, and the distribution of collagen fibril on electron microscope were assessed.

RESULTS: The transverse diameter and the count of fibroblasts of all groups increased significantly in the injected tendon compared to the non-injected tendon. However, there were no differences among all groups significantly (p<0.05). On electron micrograph, fibril diameters of injected tendon consisted of mainly smaller sizes with the intermediate sizes.

CONCLUSION: Prolotherapy enhances fibroblastic stimulation and elaboration of extracellular matrix. Short term use of NSAIDs may not have any adverse effects on tissue proliferation after prolotherapy.

 

 

 

Both 10% dextrose and Autologous serum lead to tissue regeneration in artificially created "holes" in cartilage in rabbit knee. 

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

Dr. Reeves' Notes:  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.

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.

The abstract is available below...

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. 

 

 

 

Prolotherapy & Achilles Tendon: Another Rat Study

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.

Dr. Reeves' Notes:  Dextrose 5% and 20% cause proliferation in rat Achilles tendon significantly more than a saline solution despite the same injection method and identical osmolarity. (1,100 mOsm) The 5% and 20% solutions were not significantly different from each other. This demonstrates an independent effect of dextrose separate from needling or hyperosmolarity.

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 & Department of Pathology, Eulji University School of Medicine, Korea:

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. 

 

K. Dean Reeves, M.D. is a physician and medical researcher in the area of pain caused by arthritis, chronic sprains and chronic strains. His private practice is located in the greater Kansas City area of Roeland Park, Kansas.  He collaborates in research with other locations across the country and internationally, and is licensed in the states of Kansas and Missouri.

Professional Bio & Publications             Contact

Copyright 2011-2014
Dr. K. Dean Reeves

No part of this site should be understood to be personal medical advice or instruction in how to perform injection therapy. A decision on treatment requires a good history and full examination and a knowledge of your treatment goals. Treatment decisions should be made in consultation with your personal healthcare professional and/or prolotherapist.