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Soluble Factors Released by Endogenous Viable Cells Enhance the Antioxidant and Chemoattractive Activities of Cryopreserved Amniotic Membrane.

Duan-Arnold Y, Gyurdieva A, Johnson A, Jacobstein DA, Danilkovitch A - Adv Wound Care (New Rochelle) (2015)

Bottom Line: Objective: Regulation of oxidative stress and recruitment of key cell types are activities of human amniotic membrane (hAM) that contribute to its benefits for wound treatment.The int-hAM CM showed a 202% greater antioxidant capacity than dev-hAM.Innovation and Conclusion: Int-hAM, in which all native components are preserved, including endogenous viable cells, demonstrated a significantly greater antioxidant and fibroblast and keratinocyte chemoattractive potential compared to dev-hAM, in which viable cells are destroyed.

View Article: PubMed Central - PubMed

Affiliation: Osiris Therapeutics, Inc. , Columbia, Maryland.

ABSTRACT

Objective: Regulation of oxidative stress and recruitment of key cell types are activities of human amniotic membrane (hAM) that contribute to its benefits for wound treatment. Progress in tissue preservation has led to commercialization of hAM. The majority of hAM products are devitalized with various degrees of matrix alteration. Data show the importance of hAM matrix preservation, but little is known about the advantages of retaining viable endogenous cells. In this study, we compared the antioxidant and chemoattractive properties of viable intact cryopreserved hAM (int-hAM) and devitalized cryopreserved hAM (dev-hAM) to determine the benefits of cell preservation. Approach: We evaluated the ability of int-hAM and dev-hAM to protect fibroblasts from oxidant-induced cell damage, to suppress oxidants, and to recruit fibroblasts and keratinocytes in vitro. Results: Both the int-hAM-derived conditioned medium (CM) and the int-hAM tissue rescued significantly more fibroblasts from oxidant-induced damage than dev-hAM (844% and 93% more, respectively). The int-hAM CM showed a 202% greater antioxidant capacity than dev-hAM. The int-hAM CM enhanced the recruitment of fibroblasts and normal and diseased keratinocytes to a greater extent than dev-hAM (1,555%, 315%, and 151% greater, respectively). Innovation and Conclusion: Int-hAM, in which all native components are preserved, including endogenous viable cells, demonstrated a significantly greater antioxidant and fibroblast and keratinocyte chemoattractive potential compared to dev-hAM, in which viable cells are destroyed. The release of soluble factors that protect fibroblasts from oxidative injury by hAM containing viable cells is a mechanism of hAM antioxidant activity, which is a novel finding of this study.

No MeSH data available.


Related in: MedlinePlus

Effect of hAMs on DHEK migration. The migration of DHEK obtained from patients with type 2 diabetes was induced by CM derived from viable intact cryopreserved human amniotic membrane (int-hAM) and devitalized cryopreserved human amniotic membrane (dev-hAM). DHEK were incubated with CM overnight, and migrated cells were stained with calcein AM and visualized microscopically. The area of migrated DHEK was quantified using ImageJ (NIH), and results are presented as % of Total Area/Field. Base medium was used as a negative (Neg) control, and KGM-2 was used as a positive (Pos) control. Images of migrated DHEK at 10×magnification for one representative experiment are shown in (A). Results of quantitative analysis are shown in (B). Data are presented as mean±SD of three independent microscopic fields in one representative experiment. Student's t-test was used for statistical analysis comparing int-hAM and dev-hAM. *p<0.05. CM, conditioned medium; DHEK, diseased human epidermal keratinocytes; KGM-2, keratinocyte growth medium.
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f5: Effect of hAMs on DHEK migration. The migration of DHEK obtained from patients with type 2 diabetes was induced by CM derived from viable intact cryopreserved human amniotic membrane (int-hAM) and devitalized cryopreserved human amniotic membrane (dev-hAM). DHEK were incubated with CM overnight, and migrated cells were stained with calcein AM and visualized microscopically. The area of migrated DHEK was quantified using ImageJ (NIH), and results are presented as % of Total Area/Field. Base medium was used as a negative (Neg) control, and KGM-2 was used as a positive (Pos) control. Images of migrated DHEK at 10×magnification for one representative experiment are shown in (A). Results of quantitative analysis are shown in (B). Data are presented as mean±SD of three independent microscopic fields in one representative experiment. Student's t-test was used for statistical analysis comparing int-hAM and dev-hAM. *p<0.05. CM, conditioned medium; DHEK, diseased human epidermal keratinocytes; KGM-2, keratinocyte growth medium.

Mentions: The migration of both NHEK and DHEK was also examined, respectively. DHEK are harvested from patients with type 2 diabetes. The CM derived from int-hAM enhanced the migration of both cell types relative to dev-hAM: migrated NHEK covered 11%±2% versus 3%±2% total area/field, respectively; and migrated DHEK covered 12%±1% versus 5%±1% of total area/field, respectively (Figs. 4 and 5). Cell migration induced by the int-hAM–derived CM is comparable to cell migration induced by positive control, where the migrated NHEK and DHEK covered 14%±1% and 11%±2% of total area/field, respectively.


Soluble Factors Released by Endogenous Viable Cells Enhance the Antioxidant and Chemoattractive Activities of Cryopreserved Amniotic Membrane.

Duan-Arnold Y, Gyurdieva A, Johnson A, Jacobstein DA, Danilkovitch A - Adv Wound Care (New Rochelle) (2015)

Effect of hAMs on DHEK migration. The migration of DHEK obtained from patients with type 2 diabetes was induced by CM derived from viable intact cryopreserved human amniotic membrane (int-hAM) and devitalized cryopreserved human amniotic membrane (dev-hAM). DHEK were incubated with CM overnight, and migrated cells were stained with calcein AM and visualized microscopically. The area of migrated DHEK was quantified using ImageJ (NIH), and results are presented as % of Total Area/Field. Base medium was used as a negative (Neg) control, and KGM-2 was used as a positive (Pos) control. Images of migrated DHEK at 10×magnification for one representative experiment are shown in (A). Results of quantitative analysis are shown in (B). Data are presented as mean±SD of three independent microscopic fields in one representative experiment. Student's t-test was used for statistical analysis comparing int-hAM and dev-hAM. *p<0.05. CM, conditioned medium; DHEK, diseased human epidermal keratinocytes; KGM-2, keratinocyte growth medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4440986&req=5

f5: Effect of hAMs on DHEK migration. The migration of DHEK obtained from patients with type 2 diabetes was induced by CM derived from viable intact cryopreserved human amniotic membrane (int-hAM) and devitalized cryopreserved human amniotic membrane (dev-hAM). DHEK were incubated with CM overnight, and migrated cells were stained with calcein AM and visualized microscopically. The area of migrated DHEK was quantified using ImageJ (NIH), and results are presented as % of Total Area/Field. Base medium was used as a negative (Neg) control, and KGM-2 was used as a positive (Pos) control. Images of migrated DHEK at 10×magnification for one representative experiment are shown in (A). Results of quantitative analysis are shown in (B). Data are presented as mean±SD of three independent microscopic fields in one representative experiment. Student's t-test was used for statistical analysis comparing int-hAM and dev-hAM. *p<0.05. CM, conditioned medium; DHEK, diseased human epidermal keratinocytes; KGM-2, keratinocyte growth medium.
Mentions: The migration of both NHEK and DHEK was also examined, respectively. DHEK are harvested from patients with type 2 diabetes. The CM derived from int-hAM enhanced the migration of both cell types relative to dev-hAM: migrated NHEK covered 11%±2% versus 3%±2% total area/field, respectively; and migrated DHEK covered 12%±1% versus 5%±1% of total area/field, respectively (Figs. 4 and 5). Cell migration induced by the int-hAM–derived CM is comparable to cell migration induced by positive control, where the migrated NHEK and DHEK covered 14%±1% and 11%±2% of total area/field, respectively.

Bottom Line: Objective: Regulation of oxidative stress and recruitment of key cell types are activities of human amniotic membrane (hAM) that contribute to its benefits for wound treatment.The int-hAM CM showed a 202% greater antioxidant capacity than dev-hAM.Innovation and Conclusion: Int-hAM, in which all native components are preserved, including endogenous viable cells, demonstrated a significantly greater antioxidant and fibroblast and keratinocyte chemoattractive potential compared to dev-hAM, in which viable cells are destroyed.

View Article: PubMed Central - PubMed

Affiliation: Osiris Therapeutics, Inc. , Columbia, Maryland.

ABSTRACT

Objective: Regulation of oxidative stress and recruitment of key cell types are activities of human amniotic membrane (hAM) that contribute to its benefits for wound treatment. Progress in tissue preservation has led to commercialization of hAM. The majority of hAM products are devitalized with various degrees of matrix alteration. Data show the importance of hAM matrix preservation, but little is known about the advantages of retaining viable endogenous cells. In this study, we compared the antioxidant and chemoattractive properties of viable intact cryopreserved hAM (int-hAM) and devitalized cryopreserved hAM (dev-hAM) to determine the benefits of cell preservation. Approach: We evaluated the ability of int-hAM and dev-hAM to protect fibroblasts from oxidant-induced cell damage, to suppress oxidants, and to recruit fibroblasts and keratinocytes in vitro. Results: Both the int-hAM-derived conditioned medium (CM) and the int-hAM tissue rescued significantly more fibroblasts from oxidant-induced damage than dev-hAM (844% and 93% more, respectively). The int-hAM CM showed a 202% greater antioxidant capacity than dev-hAM. The int-hAM CM enhanced the recruitment of fibroblasts and normal and diseased keratinocytes to a greater extent than dev-hAM (1,555%, 315%, and 151% greater, respectively). Innovation and Conclusion: Int-hAM, in which all native components are preserved, including endogenous viable cells, demonstrated a significantly greater antioxidant and fibroblast and keratinocyte chemoattractive potential compared to dev-hAM, in which viable cells are destroyed. The release of soluble factors that protect fibroblasts from oxidative injury by hAM containing viable cells is a mechanism of hAM antioxidant activity, which is a novel finding of this study.

No MeSH data available.


Related in: MedlinePlus