The effect of ascorbic acid and fluid flow stimulation on the mechanical properties of a tissue engineered pelvic floor repair material

Abstract

Synthetic non-degradable meshes used in pelvic floor surgery can cause serious complications such as tissue erosion. A repair material composed of an autologous oral fibroblast seeded degradable polylactic acid scaffold may be a viable alternative. The aims of this study were to investigate the effects of media supplementation with additives (ascorbic acid-2-phosphate, glycolic acid and 17-β-oestradiol) on the mechanical properties of these scaffolds. Oral fibroblasts were isolated from buccal mucosa. The effects of the three additives were initially compared in two-dimensional culture to select the most promising collagen stimulating additive. Sterile electrospun scaffolds were seeded with 500,000 oral fibroblasts and fixed in 6-well plates and subjected to ascorbic acid-2-phosphate (the best performing additive) and/or mechanical stimulation. Mechanical stimulation by fluid shear stress was induced by rocking scaffolds on a platform shaker for 1 h/day for 10 of 14 days of culture. In two-dimensional culture, ascorbic acid-2-phosphate (concentrations from 0.02 mM to 0.04 M) and glycolic acid (10 µM) led to significantly greater total collagen production, but ascorbic acid-2-phosphate at 0.03 mM produced the greatest stimulation (of the order of >100%). In three-dimensional culture, mechanical stimulation alone gave non-significant increases in stiffness and strength. Ascorbic acid-2-phosphate (0.03 mM) significantly increased collagen production in the order 280% in both static and mechanically stimulated scaffolds (p < 0.0001). There was no additional effect of mechanical stimulation. Dense collagen I fibres were observed with ascorbic acid-2-phosphate supplementation. Uniaxial tensiometry showed that strength (p < 0.01) and stiffness (p <0.05) both improved significantly. A combination of ascorbic acid-2-phosphate and mechanical stimulation led to further non-signficant increases in strength and stiffness. In conclusion, a pelvic floor repair material with improved mechanical properties can be developed by supplementing culture media with ascorbic acid-2-phosphate to increase collagen I production. Future studies will assess the change in mechanical properties after implantation in an animal model.

Keywords

Artificial tissue (biomechanics)biodegradable materials biomechanical testing/analysis scaffold development (tissue engineering)tissue culture techniques

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References

Perry

Shaw

Assassa

. An epidemiological study to establish the prevalence of urinary symptoms and felt need in the community: the Leicestershire MRC Incontinence Study. Leicestershire MRC Incontinence Study Team. J Public Health Med 2000; 22: 427–434.

Turner

. The costs of stress urinary incontinence. Health Serv J 2004; 114: suppl. 11–14 following 54.

Handa

Garrett

Hendrix

. Progression and remission of pelvic organ prolapse: a longitudinal study of menopausal women. Am J Obstet Gynecol 2004; 190: 27–32.

Hendrix

Clark

Nygaard

. Pelvic organ prolapse in the Women’s Health Initiative: gravity and gravidity. Am J Obstet Gynecol 2002; 186: 1160–1166.

Fritel

Varnoux

Zins

. Symptomatic pelvic organ prolapse at midlife, quality of life, and risk factors. Obstet Gynecol 2009; 113: 609–616.

Coyne

Kvasz

Ireland

. Urinary incontinence and its relationship to mental health and health-related quality of life in men and women in Sweden, the United Kingdom, and the United States. Eur Urol 2012; 61: 88–95.

Abdel-Fattah

Familusi

Fielding

. Primary and repeat surgical treatment for female pelvic organ prolapse and incontinence in parous women in the UK: a register linkage study. BMJ 2011; 1: e000206.

http://www.england.nhs.uk/wp-content/uploads/2013/06/e10-comp-gynae-recur-pro-urina-incon.pdf.

Davila

Drutz

Deprest

. Clinical implications of the biology of grafts: conclusions of the 2005 IUGA Grafts Roundtable. Int Urogynecol J Pelvic Floor Dysfunct 2006; 17(Suppl. 1): S51–S55.

10.

Patel

Ostergard

Sternschuss

. Polypropylene mesh and the host response. Int Urogynecol J 2012; 23: 669–679.

11.

Hung

Wen

Hung

. Tissue-engineered fascia from vaginal fibroblasts for patients needing reconstructive pelvic surgery. Int Urogynecol J 2010; 21: 1085–1093.

12.

De Filippo

Yoo

Atala

. Engineering of vaginal tissue in vivo. Tissue Eng 2003; 9: 301–306.

13.

Butler

Goldstein

Guilak

. Functional tissue engineering: the role of biomechanics. J Biomech Eng 2000; 122: 570–575.

14.

Badylak

Gilbert

Myers-Irvin

. The extracellular matrix as a biologic scaffold for tissue engineering. In: De Boer

Blitterswijk

Thomsen

. (eds) Tissue engineering. 1st ed. Oxford: Academic Press, 2008, pp.122–143.

15.

Campeau

Gorbachinsky

Badlani

. Pelvic floor disorders: linking genetic risk factors to biochemical changes. BJU Int 2011; 108: 1240–1247.

16.

Chen

DeSautel

Anderson

. Collagen synthesis is not altered in women with stress urinary incontinence. Neurourol Urodyn 2004; 23: 367–373.

17.

Chen

Wen

. Collagen metabolism and turnover in women with stress urinary incontinence and pelvic prolapse. Int Urogynecol J Pelvic Floor Dysfunct 2002; 13: 80–87; discussion 87.

18.

Moalli

Talarico

Sung

. Impact of menopause on collagen subtypes in the arcus tendineous fasciae pelvis. Am J Obstet Gynecol 2004; 190: 620–627.

19.

Reid

Luo

. Site-specific prolapse surgery. II. Vaginal paravaginal repair augmented with either synthetic mesh or remodelling xenograft. Int Urogynecol J 2011; 22: 601–609.

20.

Mangera

Bullock

Roman

. Comparison of candidate scaffolds for tissue engineering for stress urinary incontinence and pelvic organ prolapse repair. BJU Int 2013; 112: 674–685.

21.

Badylak

Kokini

Tullius

. Strength over time of a resorbable bioscaffold for body wall repair in a dog model. J Surg Res 2001; 99: 282–287.

22.

Murad

Grove

Lindberg

. Regulation of collagen synthesis by ascorbic acid. Proc Natl Acad Sci U S A 1981; 78: 2879–2882.

23.

Kim

Won

. The effect of glycolic acid on cultured human skin fibroblasts: cell proliferative effect and increased collagen synthesis. J Dermatol 1998; 25: 85–89.

24.

Moy

Howe

Moy

. Glycolic acid modulation of collagen production in human skin fibroblast cultures in vitro. Dermatol Surg 1996; 22: 439–441.

25.

Stevenson

Taylor

Meskiri

. Differing responses of human follicular and nonfollicular scalp cells in an in vitro wound healing assay: effects of estrogen on vascular endothelial growth factor secretion. Wound Repair Regen 2008; 16: 243–253.

26.

Liu

Hatch

. Effect of estrogen on cellular metabolism of the human anterior cruciate ligament. Clin Orthop Relat Res 1999; 366: 229–238.

27.

Liu

Al-Shaikh

Panossian

. Estrogen affects the cellular metabolism of the anterior cruciate ligament. A potential explanation for female athletic injury. Am J Sports Med 1997; 25: 704–709.

28.

Moehrer

Hextall

Jackson

. Oestrogens for urinary incontinence in women. Cochrane Database Syst Rev 2003; 2: CD001405.

29.

Abe

Yokoyama

. Effects of 17beta-estradiol on matrix metalloproteinase-1 synthesis by human dermal fibroblasts. Maturitas 2006; 54: 39–46.

30.

Selim

Bullock

Blackwood

. Developing biodegradable scaffolds for tissue engineering of the urethra. BJU Int 2011; 107: 296–302.

31.

Delaine-Smith

MacNeil

Reilly

. Matrix production and collagen structure are enhanced in two types of osteogenic progenitor cells by a simple fluid shear stress stimulus. Eur Cell Mater 2012; 24: 162–174.

32.

Petros

Ulmsten

Papadimitriou

. The autogenic ligament procedure: a technique for planned formation of an artificial neo-ligament. Acta Obstet Gynecol Scand Suppl 1990; 153: 43–51.

33.

Poncet

Meyer

Richard

. The expression and function of the endothelin system in contractile properties of vaginal myofibroblasts of women with uterovaginal prolapse. Am J Obstet Gynecol 2005; 192: 426–432.

34.

Ruiz-Zapata

Kerkhof

Zandieh-Doulabi

. Fibroblasts from women with pelvic organ prolapse show differential mechanoresponses depending on surface substrates. Int Urogynecol J 2013; 24: 1567–1575.

35.

Lee

Silva

Mooney

. Growth factor delivery-based tissue engineering: general approaches and a review of recent developments. J R Soc Interface 2011; 8: 153–170.

36.

Kuo

Chen

Hahn

. Collagen biosynthesis in human oral submucous fibrosis fibroblast cultures. J Dent Res 1995; 74: 1783–1788.

37.

De Landsheere

Munaut

Nusgens

. Histology of the vaginal wall in women with pelvic organ prolapse: a literature review. Int Urogynecol J 2013; 24: 2011–2020.

38.

Zong

Meyn

Moalli

. The amount and activity of active matrix metalloproteinase 13 is suppressed by estradiol and progesterone in human pelvic floor fibroblasts. Biol Reprod 2009; 80: 367–374.

39.

Moalli

Klingensmith

Meyn

. Regulation of matrix metalloproteinase expression by estrogen in fibroblasts that are derived from the pelvic floor. Am J Obstet Gynecol 2002; 187: 72–79.

40.

Mangera

Bullock

Chapple

. Are biomechanical properties predictive of the success of prostheses used in stress urinary incontinence and pelvic organ prolapse? A systematic review. Neurourol Urodyn 2012; 31: 13–21.

41.

Patel

Fine

Sandig

. Elastin biosynthesis: the missing link in tissue-engineered blood vessels. Cardiovasc Res 2006; 71: 40–49.

42.

Bye

Wang

Bullock

. Postproduction processing of electrospun fibres for tissue engineering. J Vis Exp 2012. DOI: 10.3791/4172.