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Corneal stromal cell responses to traumatic wounds and topical treatments
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-114700DOI: 10.3384/diss.diva-114700ISBN: 978-91-7519-111-9 (print)OAI: oai:DiVA.org:liu-114700DiVA: diva2:792069
Public defence
2015-03-27, Nils Holgersalen, Campus US, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2015-03-03 Created: 2015-03-03 Last updated: 2016-02-10Bibliographically approved
List of papers
1. The expression pattern of the subunit of chaperonin containing T-complex polypeptide 1 and its substrate, α-smooth muscle actin, during corneal wound healing
Open this publication in new window or tab >>The expression pattern of the subunit of chaperonin containing T-complex polypeptide 1 and its substrate, α-smooth muscle actin, during corneal wound healing
2005 (English)In: Acta Ophthalmologica Scandinavica, ISSN 1395-3907, E-ISSN 1600-0420, Vol. 83, no 5, 543-548 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: This study was designed to demonstrate the expression of the chaperonin containing T-complex polypeptide 1 (CCT) and α-smooth muscle actin (α-SMA), in normal corneas and corneas treated with ultraviolet radiation (UVR). The wound model chosen is previously characterized, the injury is mild and the cornea heals to transparency. Methods: Rabbit corneas were exposed to UVR at the dose producing keratitis. The corneas were allowed to heal for up to 5 days and the paraffin-embedded tissue specimens were double stained and examined morphologically and immunohistochemically. Expression of CCT and α-SMA genes was investigated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Results: There was a front of repopulating keratocytes that showed positive staining for α-SMA after 3 days. The α-SMA mRNA was already strongly expressed after 1 day, whereas the expression level of CCT was increased after 2 days. After 5 days the levels were decreased. By this time the stroma was partly repopulated by keratocytes. Conclusion: 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. Copyright © Acta Ophthalmol Scand 2005.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-29987 (URN)10.1111/j.1600-0420.2005.00482.x (DOI)15425 (Local ID)15425 (Archive number)15425 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13
2. Platelet Rich Plasma Prolongs Myofibroblast Accumulation in Corneal Stroma with Incisional Wound
Open this publication in new window or tab >>Platelet Rich Plasma Prolongs Myofibroblast Accumulation in Corneal Stroma with Incisional Wound
2015 (English)In: Current Eye Research, ISSN 0271-3683, E-ISSN 1460-2202, Vol. 40, no 11, 1102-1110 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: The purpose of this study was to determine whether platelet rich plasma (PRP) has an effect on corneal stromal cells in a rat model of wound healing following corneal incision. Materials and Methods: The effect of PRP on corneal wound healing in vivo was investigated in a corneal incision wound model in rats. 40 rats were wounded by deep corneal incision, and treated with either topically administered PRP (20 rats) or sodium chloride (20 rats). At 4 hours and 1, 3, and 5 days after incision, α-smooth muscle actin (α SMA), SMAD2 and SMAD3 expression and apoptosis in stromal cells were evaluated by immunohistochemistry, and IL-1β mRNA expression was evaluated by real time PCR.

Results: PRP treated corneas exhibited reduced stromal cell apoptosis at day 3 and day 5 (p = 0.038, and <0.001, respectively) relative to controls. Interleukin-1β mRNA expression, however, was unchanged in PRP treated corneas relative to controls. Topical PRP treatment resulted in a higher proportion of αSMA-positive myofibroblasts recruited to the wound site relative to control corneas. PRP did not affect activation of SMAD2 but activation of SMAD3 was significantly reduced at day 1 (p=0.001) and dramatically increased at day 5 (p=0.032).

Conclusions: 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.

Place, publisher, year, edition, pages
Taylor & Francis, 2015
Keyword
Platelet rich plasma, corneal wound healing, α-smooth muscle actin, apoptosis, keratocytes
National Category
Cell and Molecular Biology Medical Biotechnology
Identifiers
urn:nbn:se:liu:diva-114698 (URN)10.3109/02713683.2014.978478 (DOI)000369891500004 ()
Note

Funding agencies:Swedish Research Council, Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse, County Council of Ostergotland 

Vid tiden för disputation förelåg publikationen endast som manuskript

Available from: 2015-03-03 Created: 2015-03-03 Last updated: 2017-12-04Bibliographically approved
3. Topical Biglycan Modulates Stromal Cell Apoptosis in Corneal Incisional Wound Model
Open this publication in new window or tab >>Topical Biglycan Modulates Stromal Cell Apoptosis in Corneal Incisional Wound Model
2015 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Purpose: The purpose of this study was to determine whether exogenous topicallyapplied biglycan has an effect on corneal stromal cells during wound healing.

Methods: Enzyme-linked immunosorbent assay (ELISA) was used to determine the effect of biglycan on cell survival in vitro following IL-1β induced cell death. In a corneal incisional wound model, 40 rats were wounded and treated with either topically administered biglycan or sodium chloride (sham control). At 4 hours and 1, 2, and 5 days after incision, α-smooth muscle actin (SMA) expression and apoptosis in stromal cells were evaluated by immunohistochemistry.

Results: In vitro, biglycan significantly enhanced IL-1β-induced apoptosis of myofibroblasts (p = 0.038), but not corneal fibroblasts. Biglycan treated corneas exhibited reduced stromal cell apoptosis at 4 hours, day 1 and day 5 (p = 0.012, 0.040, and 0.048, respectively) and increased apoptosis at day 3 (p = 0.003) relative to controls. In wounded corneas, biglycan appeared to promote early accumulation of myofibroblasts and initiate an earlier subsequent apoptosis of these cells, relative to controls.

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

Keyword
Corneal wound healing; biglycan; keratocytes; IL-1β; α-SMA
National Category
Cell and Molecular Biology Medical Biotechnology
Identifiers
urn:nbn:se:liu:diva-114697 (URN)
Available from: 2015-03-03 Created: 2015-03-03 Last updated: 2016-04-01
4. Enhanced Regeneration of Corneal Tissue Via a Bioengineered Collagen Construct Implanted by a Nondisruptive Surgical Technique
Open this publication in new window or tab >>Enhanced Regeneration of Corneal Tissue Via a Bioengineered Collagen Construct Implanted by a Nondisruptive Surgical Technique
Show others...
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
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-114699 (URN)10.1089/ten.tea.2014.0562 (DOI)000350549500025 ()25412075 (PubMedID)
Available from: 2015-03-03 Created: 2015-03-03 Last updated: 2017-12-04Bibliographically approved

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