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Enhanced Regeneration of Corneal Tissue Via a Bioengineered Collagen Construct Implanted by a Nondisruptive Surgical Technique
Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Ophthalmology in Linköping. Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Biomedical Engineering, Biomedical Instrumentation. Linköping University, Faculty of Science & Engineering. Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Medicine and Health Sciences. LinkoCare Life Sciences AB, Linköping, Sweden.ORCID iD: 0000-0001-6024-4144
University of Debrecen, Debrecen, Hungary; University of Szeged, Szeged, Hungary.
University of Debrecen, Debrecen, Hungary; University of Szeged, Szeged, Hungary.
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2015 (English)In: Tissue Engineering. Part A, ISSN 1937-3341, E-ISSN 1937-335X, Vol. 21, no 5-6, 1116-1130 p.Article in journal (Refereed) Published
Abstract [en]

Severe shortage of donor corneas for transplantation, particularly in developing countries, has prompted the advancement of bioengineered tissue alternatives. Bioengineered corneas that can withstand transplantation while maintaining transparency and compatibility with host cells, and that are additionally amenable to standardized low-cost mass production are sought. In this study, a bioengineered porcine construct (BPC) was developed to function as a biodegradable scaffold to promote corneal stromal regeneration by host cells. Using high-purity medical-grade type I collagen, high 18% collagen content and optimized EDC-NHS cross-linker ratio, BPCs were fabricated into hydrogel corneal implants with over 90% transparency and four-fold increase in strength and stiffness compared with previous versions. Remarkably, optical transparency was achieved despite the absence of collagen fibril organization at the nanoscale. In vitro testing indicated that BPC supported confluent human epithelial and stromal-derived mesenchymal stem cell populations. With a novel femtosecond laser-assisted corneal surgical model in rabbits, cell-free BPCs were implanted in vivo in the corneal stroma of 10 rabbits over an 8-week period. In vivo, transparency of implanted corneas was maintained throughout the postoperative period, while healing occurred rapidly without inflammation and without the use of postoperative steroids. BPC implants had a 100% retention rate at 8 weeks, when host stromal cells began to migrate into implants. Direct histochemical evidence of stromal tissue regeneration was observed by means of migrated host cells producing new collagen from within the implants. This study indicates that a cost-effective BPC extracellular matrix equivalent can incorporate cells passively to initiate regenerative healing of the corneal stroma, and is compatible with human stem or organ-specific cells for future therapeutic applications as a stromal replacement for treating blinding disorders of the cornea.

Place, publisher, year, edition, pages
Mary Ann Liebert, 2015. Vol. 21, no 5-6, 1116-1130 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-114699DOI: 10.1089/ten.tea.2014.0562ISI: 000350549500025PubMedID: 25412075OAI: oai:DiVA.org:liu-114699DiVA: diva2:792063
Available from: 2015-03-03 Created: 2015-03-03 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Corneal stromal cell responses to traumatic wounds and topical treatments
Open this publication in new window or tab >>Corneal stromal cell responses to traumatic wounds and topical treatments
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background. The cornea has unique anatomic, cellular, molecular, and functional features that lead to important mechanistic differences in the process of repair in comparison with what occurs in skin and other organs. The first observable stromal response in corneal wound healing is keratocyte apoptosis. Shortly thereafter, remaining keratocytes in adjacent areas obtain a fibroblastic phenotype and begin to proliferate and to migrate, transforming into myofibroblasts, a phenotype associated with remodeling of stromal collagen. Return to normalcy following wound healing includes elimination of myofibroblasts and restoration of the quiescent state of the keratocytes. Often, however, a wound healing response results in the persistence of myofibroblasts and their subsequent production of fibrous scar tissue.

Aims. The overall aim is to understand the role of keratocytes, and their phenotypic variations in a cornea subjected to various types of trauma or treatments. More specific aims are to define expression pattern of alpha-smooth muscle actin (α-SMA) and chaperonin containing T-complex polypeptide 1 (CCT) in ultraviolet radiation wound model, to evaluate the effect of biglycan and platelet rich plasma (PRP) treatment during wound healing after corneal incision, and to characterize the structure of the bioengineered porcine construct and its interaction with stromal cells after implantation.

Methods. CCT and α-SMA expression level was evaluated by reverse transcription polymerase chain reaction (RT-PCR) in rabbit corneas subjected to ultraviolet radiation (UVR). Effect of biglycan and PRP on keratocyte phenotype and survival was evaluated by immunohistochemistry, and real time PCR using rat corneas after  incisional wounding. Bioengineered porcine construct (BPC) was implanted into rabbit corneas using femtosecond laser-enabled intrastromal keratoplasty (FLISK) and characterized by means of immunohistochemistry, electron microscopy, and in vivo confocal microscopy (IVCM).

Results and conclusions. In a mild wound, the expression of α-SMA mRNA is followed by expression of mRNA of at least one subunit of the complex folding α-SMA. At protein level, α-SMA is detected in the front line of repopulating keratocytes. Expression levels for both mRNAs decline as the stroma repopulation process progresses.

Biglycan appears to accelerate corneal wound healing in vivo by modulating myofibroblast apoptosis, resulting in removal of myofibroblasts that may otherwise compromise corneal transparency.

PRP treatment resulted in suppressed stromal cell apoptosis followed by SMAD3 activation and a greater proportion of myofibroblasts present at the wound site. Suppression of stromal cell apoptosis after corneal wounding by use of a growth factor rich formulation may lead to myofibroblast accumulation by modulation of the TGF-β pathway.

A cost-effective BPC extracellular matrix equivalent can incorporate cells passively to initiate normal regenerative healing of the corneal stroma.

Taken together, results present an interesting possibility to combine BPC implantation and topical biglycan treatment to improve surgical outcome in future studies.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. 88 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1451
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-114700 (URN)10.3384/diss.diva-114700 (DOI)978-91-7519-111-9 (ISBN)
Public defence
2015-03-27, Nils Holgersalen, Campus US, Linköping, 13:00 (English)
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Available from: 2015-03-03 Created: 2015-03-03 Last updated: 2016-02-10Bibliographically approved

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Koulikovska, MarinaRafat, MehrdadFagerholm, PerLagali, Neil

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Department of Ophthalmology in LinköpingDivision of Neuro and Inflammation ScienceFaculty of Medicine and Health SciencesBiomedical InstrumentationFaculty of Science & EngineeringDepartment of Clinical and Experimental MedicineDepartment of Ophthalmology in Linköping
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Tissue Engineering. Part A
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