In Vitro Research

In Vitro Research
Primarily on Human Cells

Dr. Reeves' Summary: Many studies have shown dextrose effects on growth factors. These are found in diabetic research due to the large interest in why dextrose elevations in diabetics causes unwanted growth of cells in many areas such as blood vessels in the eye, lining cells in arteries, glomerular cells in the kidney, etc. Those who consider dextrose a placebo are correct when it is taken by mouth but wrong when it is administered by injection, bypassing the effects of digestion. In prolotherapy the intent is not to elevate dextrose throughout the body. Rather the intent is to localize dextrose by injection to create a response at the site of injection only. Human cell studies are not including cartilage and ligament and tendon cells harvested from humans, such as patients during knee and hip replacements, enabling a number of studies to be done.


Hyaluronic Acid (Joint Lubricant) Production Increases With Exposure to Dextrose or Glucosamine (2008)

Uitterlinden EJ; Koevoet JL; Verkoelen CF; Bierma-Zeinstra SM; Jahr H; Weinans H; Verhaar JA; van Osch GJ. Glucosamine increases hyaluronic acid production in human osteoarthritic synovium explants [epub ahead of print] BMC Musculoskelet Disord (), Sep 11 2008, 9(1) p120.

The abstract is available below...

BACKGROUND: Glucosamine (GlcN) used by patients with osteoarthritis was demonstrated to reduce pain, but the working mechanism is still not clear. Viscosupplementation with hyaluronic acid (HA) is also described to reduce pain in osteoarthritis. The synthesis of HA requires GlcN as one of its main building blocks. We therefore hypothesized that addition of GlcN might increase HA production by synovium tissue.

METHODS: Human osteoarthritic synovium explants were obtained at total knee surgery and pre-cultured for 1 day. The experimental conditions consisted of a 2 days continuation of the culture with addition of N-Acetyl-glucosamine (GlcN-Ac; 5 mM), glucosamine-hydrochloride (GlcN-HCl; 0.5 and 5 mM), glucose (Gluc; 0.5 and 5 mM). Hereafter HA production was measured in culture medium supernatant using an enzyme-linked binding protein assay. Real time RT-PCR was performed for hyaluronic acid synthase (HAS) 1, 2 and 3 on RNA isolated from the explants. RESULTS: 0.5 mM and 5 mM GlcN-HCl significantly increased HA production compared to control (approximately 2 - 4-fold), whereas GlcN-Ac had no significant effect. Addition of 5 mM Gluc also increased HA production (approximately 2-fold), but 0.5 mM Gluc did not. Gene expression of the HA forming enzymes HAS 1, 2 and 3 was not altered by the addition of GlcN or Gluc.

CONCLUSIONS: Our data suggest that exogenous GlcN can increase HA production by synovium tissue and is more effective at lower concentrations than Gluc. This might indicate that GlcN exerts its potential analgesic properties through stimulation of synovial HA production.


Reeves et al 2008: Growth Factor Production Occurs Promptly After Exposure By A Variety of Body Cells to Dextrose and Thus Would Be Expected to Increase Hyaluronic Acid Production as Well.

Reeves KD Topol GA Fullerton BD Evidence-based regenerative injection therapy (prolotherapy) in sports medicine. In Seidenberg PH, Beutler PI. (Eds). The Sports Medicine Resource Manual. Saunders (Elsevier); 2008: 611-619.

Dr. Reeves' Notes:  Cells often produce growth factors which cause their own repair and multiplication. Ligament and tendon and cartilage cells are stimulated to produce the key growth factors for repair and replication by exposure to as little at 0.6% dextrose. Note also that growth factors for bone ARE NOT stimulated by dextrose exposure. Therefore there is no concern about causing spur formation of bony responses to dextrose.


Effects of bupivacaine on articular chondrocytes (2008)

Chu et al 2008 Exposure to bupivacaine damages cartilage cells. Chu CR; Izzo NJ; Coyle CH; Papas NE; Logar A. The in vitro effects of bupivacaine on articular chondrocytes J Bone Joint Surg Br (England), Jun 2008, 90(6) p814-20

The abstract is available below...

Summary: We have studied the effects of bupivacaine on human and bovine articular chondrocytes in vitro. Time-lapse confocal microscopy of human articular chondrocytes showed > 95% cellular death after exposure to 0.5% bupivacaine for 30 minutes. Human and bovine chondrocytes exposed to 0.25% bupivacaine had a time-dependent reduction in viability, with longer exposure times resulting in higher cytotoxicity. Cellular death continued even after removal of 0.25% bupivacaine. After exposure to 0.25% bupivacaine for 15 minutes, flow cytometry showed bovine chondrocyte viability to be 41% of saline control after seven days. After exposure to 0.125% bupivacaine for up to 60 minutes, the viability of both bovine and human chondrocytes was similar to that of control groups. These data show that prolonged exposure 0.5% and 0.25% bupivacaine solutions are potentially chondrotoxic.


Hyaluronic Acid (Joint Lubricant) Production Increases With Exposure to Platelet Released Growth Factors (2007)

Growth Factors From Platelets Stimulate Hyaluronic Acid and Other Growth Factors As Well. E.Anitua, M. Sánchez, A. T. Nurden, M. M. Zalduendo, M. de la Fuente, J. Azofra and I. Andía Platelet-released growth factors enhance the secretion of hyaluronic acid and induce hepatocyte growth factor production by synovial fibroblasts from arthritic patients Rheumatology 2007 46(12)

The abstract is available below...

OBJECTIVES.  Autologous platelet-secreted growth factors (GFs) may have therapeutic effects in osteoarthritis (OA) capsular joints via multiple mechanisms. Our aim was to examine the effect of a platelet-derived preparation rich in growth factors (PRGFs) in OA synovial cell biology.

METHODS. Synovial cells were isolated from 10 osteoarthritic patients and cultured in serum-free media (basal conditions) and exposed to either a platelet-poor preparation or PRGF for 72 h. Cells activated with interleukin-1ß in several events relevant to joint homeostasis including (I) hyaluronic acid (HA) secretion, (ii) the balance between metalloproteinase-1, -3 and -13 (MMP-1, MMP-3 and MMP-13) and tissue inhibitor-1 (TIMP-1) and (iii) the secretion of transforming growth factor-ß1(TGF-ß1), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), were all assessed.

RESULTS.  PRGF significantly enhanced HA secretion compared with platelet-poor preparations, P < 0.05; at the same time release of TIMP-1, MMP-1, MMP-3 and MMP-13 were not affected. An increased HGF production was observed (P < 0.05) but VEGF and TGF-ß1 levels remained unchanged. PRGF significantly enhanced the secretion of HA induced by IL-1ß activation, P < 0.05, but it did not modify the IL-1ß-induced rise in MMP-1, MMP-3 and VEGF. In contrast, PRGF-induced HGF production was abolished by the presence of IL-1ß during PRGF treatment, P < 0.05.

CONCLUSIONS.  Intra-articular administration of PRGF might be beneficial in restoring HA concentration and switching angiogenesis to a more balanced status but does not halt the effects of IL-1ß on synovial cells.


Effects of lidocaine on bovine articular chondrocytes (2007)

Karpie JC; Chu CR Lidocaine exhibits dose- and time-dependent cytotoxic effects on bovine articular chondrocytes in vitro. Am J Sports Med (United States), Oct 2007, 35(10) p1621-7

The abstract is available below...

BACKGROUND: Intra-articular lidocaine is commonly used. PURPOSE: This study was conducted to determine whether short-term exposures to 1% and 2% lidocaine are toxic to articular chondrocytes, whether this is due to pH, and whether an intact articular surface is protective.

STUDY DESIGN: Controlled laboratory study.

METHODS: Fresh bovine articular chondrocytes in alginate bead cultures were treated with 1% or 2% lidocaine or buffered saline (pH 7.4, 7.0, and 5.0) for 15, 30, or 60 minutes. Chondrocytes were then analyzed for viability by flow cytometry 1 hour, 1 day, and 1 week later. Bovine osteochondral cores with and without the superficial 1 mm of cartilage removed were submerged in either 0.9% saline (pH 7.4) or in 1% or 2% lidocaine for 30 minutes and assessed for viability using fluorescent microscopy.

RESULTS: Chondrocyte viability decreased after just 15-minute exposures to 1% lidocaine. Longer exposures to 1% and 2% lidocaine further reduced chondrocyte viability. Chondrotoxicity of 2% lidocaine was greater than 1% lidocaine. There was no difference in chondrocyte viability after exposures to saline solutions of pH 7.4, 7.0, or 5.0. An intact articular surface did not affect lidocaine's chondrotoxic effects.

CONCLUSION: Results show dose- and time-dependent cytotoxic effects of lidocaine on bovine articular chondrocytes. Reduction of pH alone did not decrease chondrocyte viability, and the intact articular surface was not protective.

CLINICAL RELEVANCE: Although lidocaine chondrotoxicity was less than previously reported with bupivacaine, these observations suggest that local anesthetics as a class of drugs may negatively affect articular cartilage.


Injections and repairing retina structural damage (2007)

Injection of benzyl alcohol may repair severe structural damage to the retina when injected in rabbit eyes. Macky TA, Helmy D, El Shazly N. Retinal toxicity of triamcinolone's vehicle (benzyl alcohol): an electrophysiologic and electron microscopic study. Graefes Arch Clin Exp Ophthalmol 2007; 245(6): 817-824 

The abstract is available below...

PURPOSE: To assess retinal toxicity of the vehicle of triamcinolone, benzyl alcohol (BA), when injected into the vitreous cavity of rabbits.

METHODS: This prospective comparative experimental study included 24 pigmented rabbits assigned into two groups: group 1 (experimental, n = 12) received intravitreal 0.1 ml of BA, and group 2 (control, n = 12) received intravitreal 0.1 ml of balanced salt solution (BSS); all injections were done in the right eyes. Clinical examinations [slit lamp biomicroscopy, indirect ophthalmoloscopy, and three intraocular pressure (IOP) measurements] were done on both eyes before injection, at 1 and 3 h post injection, together with electroretinograms (ERGs) at 3 days, 1, 2, 4, and 6 weeks following injections. Three rabbits from each group were euthanized at 1, 2, 4, or 6 weeks and eyes were sent for light and electron microscopic examination for quantitative morphometric measurements.

RESULTS: The mean amplitudes of the a and b waves of the BA-injected eyes were 6.42 +/- 9.02 microv and 11.18 +/- 15.18 microv at 3 days, respectively, which were significantly reduced compared with the BSS-injected eyes (30.87 +/- 8.22 microv and 57.90 +/- 13.38 microv, respectively; P < 0.01 t-test) and the non-injected contralateral eyes (36.20 +/- 7.85 microv and 64.10 +/- 9.36 microv, respectively; P < 0.01 t-test). These ERG responses continued to be significantly reduced in the BA-injected eyes (P < 0.01 t-test) throughout the study period. The mean ganglion cell count was significantly reduced (P < 0.005 t-test) in the BA-injected eyes (8.42 +/- 2.4) compared with the BSS- and non-injected eyes (16.42 +/- 3.9 and 16.5 +/- 4.2, respectively). The mean thicknesses of the inner nuclear layer (INL) and outer nuclear layer (ONL) were significantly reduced (P < 0.005 t-test) in the BA-injected eyes (3.78 +/- 0.96 microm and 11.77 +/- 1.29 microm, respectively) compared with the BSS- (6.1 +/- 0.92 microm and 21.82 +/- 0.95 microm, respectively) and non-injected eyes (7.05 +/- 1.9 microm and 22.49 +/- 1.01 microm, respectively). Electron microscopy showed moderate to severe intracellular changes in the ganglion cell layer, INL, ONL, and photoreceptor layer at 6 weeks in BA-injected eyes, with no significant changes in BSS-injected eye. There was no significant rise in the IOP or clinical evidence of increased lens density during the study period in any of the eyes.

CONCLUSIONS: Triamcinolone acetonide's vehicle, BA, produced severe ERG and structural damage to the retina when injected intravitreally.


Morrison et al 2006: Injection of preservative (benzyl alcohol) may damage retinal cells.

Morrison VL; Koh HJ; Cheng L; Bessho K; Davidson MC; Freeman WR A Intravitreal toxicity of the kenalog vehicle (benzyl alcohol) in rabbits. Retina (United States), Mar 2006, 26(3) p339-44.

Dr. Reeves' Notes:  Work with live cartilage cells has not thus far included work with benzyl alcohol directly but studies related to eyes are very important to consider. The eye fluid (vitreous) is a fluid area with a lot of hyaluronic acid, a bit like a synovial joint. The following studies show substantial harm to the retina in animals injected in the eye with steroids including the preservative benzyl alcohol . This needs to be shown in other studies of course and specifically on cartilage cells but it raises a word of caution about the merit of "making our solutions safer" by putting preservative in them.

The abstract is available below...

PURPOSE: To test the toxicity of intravitreal injections of benzyl alcohol.

METHODS: Nine New Zealand rabbits were injected with either a control or a test article at elevating concentrations. The test article was benzyl alcohol calculated to give final injected concentrations of 0.0073%, 0.022%, 0.073%, 0.222%, and 0.733% benzyl alcohol. The 0.022% concentration corresponds to the concentration of benzyl alcohol in human eyes when 0.1 mL of commercial Kenalog (Bristol-Myers Squibb, Princeton, NJ) is used. Baseline examination of the rabbits was performed along with postinjection examinations on days 1, 3, 7, and 14. The eyes were enucleated and examined by light and electron microscopic examinations.

RESULTS: Eyes injected with benzyl alcohol concentrations of 0.073%, 0.222%, and 0.733% displayed changes in the outer retina including loss of, and shortening of, outer segments and photoreceptors.

CONCLUSIONS: Benzyl alcohol at concentrations modestly higher than what is present in commercial Kenalog is toxic to the rabbit eye. This has been shown in other organ systems. If commercial preserved Kenalog is to be used clinically, decanting the supernatant or using other means to remove the benzyl alcohol may be considered, especially if a volume of >0.1 mL of solution is used. We hypothesize that the noninfectious inflammation seen clinically after Kenalog injection is due to the presence of a toxic preservative at unsafe concentrations.


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.

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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.