Send to

Choose Destination
Clin Orthop Relat Res. 2017 Aug;475(8):2117-2127. doi: 10.1007/s11999-017-5370-1. Epub 2017 Apr 27.

Prolotherapy Induces an Inflammatory Response in Human Tenocytes In Vitro.

Author information

Department of Biomedical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ, 08854, USA.
Edward Via Virginia College of Osteopathic Medicine, Blacksburg, VA, USA.
Experimental Trauma Surgery, Department of Trauma Surgery, University Regensburg Medical Centre, Regensburg, Germany.
Department of Tissue Regeneration, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands.
The Center for Neuroscience and Cell Biology, Stem Cells and Drug Screening Lab, University of Coimbra, Largo Marquês de Pombal Coimbra, Portugal.
Department of Biomedical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ, 08854, USA.



Proliferative therapy, or prolotherapy, is a controversial treatment method for many connective tissue injuries and disorders. It involves the injection of a proliferant, or irritant solution, into the site of injury, which causes small-scale cell death. This therapeutic trauma is theorized to initiate the body's wound-healing cascade, perhaps leading to tissue repair. The immediate effects of many of these proliferants are poorly characterized, as are the cellular responses to them; here, we sought to evaluate the immediate effects of two common proliferants (dextrose and P2G, a combination of phenol, glucose, and glycerin) on the cellular response of human tenocytes, and begin to explicate the mechanisms with which each proliferant functions.


We asked: What are the effects of treating cultured tenocytes with proliferative treatment agents on their (1) cellular metabolic activity, (2) RNA expression, (3) protein secretion, and (4) cell migration?


Using human hamstring and Achilles tendon cells, we attempted to answer our research questions. We used a colorimetric metabolic assay to assess the effect of dextrose and P2G proliferant treatment on cell mitochondrial activity compared with nontreated tenocytes. Next, using quantitative PCR, ELISA, and a reporter cell line, we assessed the expression of several key markers involved in tendon development and inflammation. In addition, we used a scratch wound-healing assay to evaluate the effect of proliferant treatment on tenocyte migration.


Results showed that exposure to both solutions led to decreased metabolic activity of tenocytes, with P2G having the more pronounced effect (75% ± 7% versus 95% ± 7% of untreated control cell metabolic levels) (ANOVA; p < 0.01; mean difference, 0.202; 95% CI, 0.052-0.35). Next, gene expression analysis confirmed that treatment led to the upregulation of key proinflammatory markers including interleukin-8 and cyclooxygenase-2 and downregulation of the matrix marker collagen type I. Furthermore, using a reporter cell line for transforming growth factor-β (TGF-β), a prominent antiinflammatory marker, we showed that treatments led to decreased TGF-β bioactivity. Analysis of soluble proteins using ELISA revealed elevated levels of soluble prostaglandin E2 (PGE2), a prominent inducer of inflammation. Finally, both solutions led to decreased cellular migration in the tenocytes.


Taken together, these results suggest that prolotherapy, more so with P2G, may work by decreasing cellular function and eliciting an inflammatory response in tenocytes. Additional studies are needed to confirm the cellular signaling mechanisms involved and the resulting immediate response in vivo.


If these preliminary in vitro findings can be confirmed in an in vivo model, they may provide clues for a possible cellular mechanism of a common alternative treatment method currently used for certain soft tissue injuries.

[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Wolters Kluwer Icon for PubMed Central
Loading ...
Support Center