BASIC INVESTIGATION Transparency in a Fibrin and Fibrin–Agarose Corneal Stroma Substitute Generated by Tissue Engineering Juan de la Cruz Cardona, PhD,* Ana-Maria Ionescu, MSc,* Ricardo Go ´mez-Sotomayor, MSc,† Miguel Gonza ´lez-Andrades, MD,† Antonio Campos, MD, PhD,† Miguel Alaminos, MD, PhD,† and Mar´ ıa del Mar Pe ´rez, PhD* Purpose: To examine the transparency characteristics at different times of development in the culture of 2 different types of human corneal stroma substitutes generated by tissue engineering using human fibrin or human fibrin and 0.1% agarose, with human keratocytes entrapped within. Methods: The transparency of these artificial corneal stromas was analyzed after 1, 7, 14, 21, and 28 days of development in culture by determining the scattering and absorption coefficients from the spectral reflectance data of each sample using the Kubelka–Munk equations. Results: The scattering coefficient of both types of bioengineered tissues tended to increase with culture time and wavelength until 550 nm, whereby a slight decrease was observed for longer wavelengths. In general, the spectral distribution of the Kubelka–Munk scattering coefficient of the fibrin–agarose corneal constructs was more stable than that of the fibrin constructs. The absorption coefficient of the human fibrin and fibrin–agarose corneal substitutes tended to decrease with increasing wavelength, and their absolute values were higher for fibrin–agarose than for fibrin scaffolds, especially for short wave- lengths. In addition, the spectral transmittance behavior of both types of tissue analyzed was similar to the ones of the theoretical and control corneas, with absolute values above 90% for all wavelengths at 28 days of development. Conclusions: The transparency, scattering, and absorption of both fibrin and fibrin–agarose corneal stroma substitutes indicate that these new biomaterials could be adequate for clinical use. Key Words: transparency, cornea, fibrin–agarose, tissue engineering (Cornea 2011;30:1428–1435) S ight has been a primary factor of advantage in natural selection and evolution, thereby conferring great impor- tance to the maintenance of corneal transparency. 1 The cornea’s primary physiological functions are transmission of incident light, refraction, and provision of protection to intraocular structures from trauma and pathogens. 2 More than 60% of the total refractive power of the eye is attributed to the cornea, making it vital to focusing light onto the retina for visual processing. 3 For optimal vision, the cornea must efficiently transmit incident light by maintaining its transparency. When trans- parency of a native cornea cannot be maintained at a functional level for the patient, corneal transplantation is often the next intervention. Once transplanted, the major cause of corneal graft failure is allograft rejection. In this context, several researchers have focused their efforts on the development of an autologous artificial substitute of the human cornea by tissue engineering, 4–8 reducing in this way the risk of rejection. Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences to the development of biological substitutes that restore, maintain, or improve tissue function or even a whole organ. 9 These techniques make it possible to develop several types of bioengineered tissues that could be used for clinical purposes. 10–13 Because native tissues are 3-dimensional structures, construction of efficient tissue substitutes depends strongly on the use of scaffolds, the structure of which must resemble the extracellular matrix of the native tissues. However, the creation of 3-dimensional scaffolds that mimic the structure of the human cornea is a major bioengineering challenge. 14 So far, several biomate- rials have been used as biological substitutes of the corneal stroma, including collagen, 14–16 chitosan, 17 polyglycolic acid, 18 and fibrin. 19 In addition, different methods have been developed by tissue engineering principles for the generation of corneal replacements, such as the method of transplantation involving a carrier-free cell sheet by exploiting temperature- responsive culture surfaces, as proposed by Nishida et al. 20 They reported good results of ocular surface reconstruction with the use of cultured, autologous, oral mucosal epithelial cells and carrier-free tissue-replacement sheets. Recently, a novel biomaterial based on a mixture of human fibrin and agarose that allowed the in vitro development of substitutes for the rabbit 4 and the human 5 cornea was designed. Although some optical parameters such as the absorbance, absorption coefficient, and transmittance of these new Received for publication May 26, 2010; revision received December 27, 2010; accepted March 3, 2010. From the *Department of Optics, Faculty of Sciences, and †Tissue Engineering Group, Department of Histology, Faculty of Medicine, University of Granada, Granada, Spain. Supported by the Spanish Plan Nacional de Investigacio ´n Cient´ ıfica, Desarrollo e Innovacio ´n Tecnolo ´gica (I+D+I), the Instituto de Salud Carlos III (ISCIII)—Subdireccio ´ n General de Evaluacio ´ n y Fomento de la Investigacio ´n (FIS PI08/614), and Proyecto MAT2009-09795 from Ministerio de Ciencia e Innovacio ´n. The authors state that they have no proprietary interest in the products named in this article. Reprints: Mar´ ıa del Mar Pe ´rez, Department of Optics, Faculty of Sciences, Campus Fuentenueva s/n, University of Granada, Granada 18071, Spain (e-mail: mmperez@ugr.es). Copyright Ó 2011 by Lippincott Williams & Wilkins 1428 | www.corneajrnl.com Cornea  Volume 30, Number 12, December 2011