0
Original Investigation |

Tissue Engineering for In Vitro Analysis of Matrix Metalloproteinases in the Pathogenesis of Keloid Lesions

Hanwei Li, PhD1; Zayna Nahas, MD1; Felicia Feng, BS1; Jennifer H. Elisseeff, PhD1; Kofi Boahene, MD2
[+] Author Affiliations
1Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, Maryland
2Department of Otolaryngology–Head and Neck Surgery, The Johns Hopkins University, Baltimore, Maryland
JAMA Facial Plast Surg. 2013;15(6):448-456. doi:10.1001/jamafacial.2013.1211.
Text Size: A A A
Published online

Importance  Keloid lesions form because of alterations in the mechanisms that govern cutaneous wound healing. Although matrix metalloproteinases (MMPs) have been implicated in keloid pathophysiology, many questions still remain about their involvement. Our incomplete understanding of keloid pathophysiology has led to high recurrence rates in current treatments. No reliable animal model is available for studying keloids.

Objective  To gain a better understanding of the disease mechanisms involved in keloid lesions in the hopes of identifying therapeutic options.

Design  Fibroblasts derived from keloid tissue were incorporated in either Matrigel or polyethylene glycol diacrylate mixed with type I collagen to create 3-dimensional models to investigate the role MMPs play in keloid formation. The MMP gene expressions were also compared between fibroblasts isolated from different sites within the same keloid lesion.

Setting  The Johns Hopkins School of Medicine, Baltimore, Maryland.

Participants  Keloid fibroblasts were received from the Baylor College of Medicine, and additional keloid fibroblasts were enzymatically isolated from the dermal layer of lesions removed from consenting patients at The Johns Hopkins Hospital.

Results  In the Matrigel system, MMP9 and MMP13 were observed to be significantly upregulated in keloid fibroblasts. The addition of decorin resulted in a significant decrease of type I collagen and MMP1, MMP9, and MMP13 gene expressions from keloid fibroblasts. Higher MMP gene expressions were observed in fibroblasts isolated from the margins of the original keloid wound.

Conclusions and Relevance  MMP9 and MMP13 are expressed significantly more in keloid-derived cells, thus making them 2 potential targets for disease modification. Molecules that target organization of the lesion’s matrix can be beneficial in downregulating increased markers during the disease. In addition, heterogeneity is observed with the varying MMP gene expressions from site-specific fibroblasts within the same keloid lesion.

Figures in this Article

Sign In to Access Full Content

Don't have Access?

Register and get free email Table of Contents alerts, saved searches, PowerPoint downloads, CME quizzes, and more

Subscribe for full-text access to content from 1998 forward and a host of useful features

Activate your current subscription (AMA members and current subscribers)

Purchase Online Access to this article for 24 hours

Figures

Place holder to copy figure label and caption
Figure 1.
Type I Bovine Collagen In Vitro Model

Letters indicate statistical significance from HS27 at corresponding time point (P < .05e and P < .001d); plus signs, statistical significance from day 2 of same cell type (P < .05c, P < .01a, and P < .001b). A, Schematic of type I bovine collagen in vitro model. Polyethylene glycol diacrylate (PEGDA) was mixed with type I collagen fibrils before encapsulation of fibroblasts (HS27 and keloid fibroblasts) through UV photopolymerization. B, Reverse transcription–polymerase chain reaction demonstrated that keloid fibroblasts had increased type I collagen gene expression compared with normal fibroblasts. C, DNA quantification normalized to dry weight indicated decreasing trend over time. D, Glycosaminoglycan (GAG) content by both fibroblast types normalized to DNA demonstrated increased GAG production from keloid fibroblasts. E, Total collagen content by both cell types normalized to DNA also demonstrated increased collagen content from keloid fibroblasts.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.
Reverse Transcription–Polymerase Chain Reaction of MMP Gene Expressions From Normal and Keloid Fibroblasts in the PEG-Col1 Model

No obvious differences in trends were observed between HS27 and keloid fibroblasts. β-Actin was the housekeeping gene.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.
Matrigel In Vitro Model

Letters indicate statistical significance from HS27 at corresponding time point (P < .05c, P < .01e, and P < .001b); plus signs, statistical significance from day 1 of same cell type (P < .05a, P < .01d, and P < .001f). A, Schematic of 3-dimensional Matrigel constructs. B, Reverse transcription–polymerase chain reaction of type I collagen from both HS27 and keloid fibroblasts after culture in Matrigel demonstrates the expected higher gene expression from keloid fibroblasts. C, DNA content decreased in both fibroblast types as time increased. D, Glycosaminoglycan (GAG) content was more significantly produced from keloid fibroblasts than HS27, resulting in a time-dependent increase. E, Total collagen content normalized to DNA also demonstrated more extracellular matrix production from keloid fibroblasts than HS27.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.
Reverse Transcription–Polymerase Chain Reaction of MMP Gene Expressions From Normal and Keloid Fibroblasts After In Vitro 3-Dimensional Culture in Matrigel

Significant differences were observed in MMP9 and MMP13 gene expressions between HS27 and keloid fibroblasts. β-Actin was the housekeeping gene.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 5.
Effect of Decorin on Keloid Fibroblasts in the Matrigel Model

Letters indicate statistical significance from HS27 at corresponding time point (P < .01b); plus signs, statistical significance from day 7 of same cell type (P < .001a). A, DNA quantification normalized to respective dry weights indicated decrease in both conditions as time increased. B, Glycosaminoglycan (GAG) content normalized to DNA demonstrated that the presence of decorin prevented an increase in the matrix component production. C, Total collagen content normalized to DNA demonstrated a similar trend as the GAG data, although not statistically significant. D, Reverse transcription–polymerase chain reaction of type I collagen and MMP genes that were affected by the presence of decorin administered to keloid fibroblasts. Significant downregulations in type I collagen, MMP1, and MMP13 were observed at day 21, whereas MMP9 retained the same expression as day 7 and was prevented from being upregulated.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 6.
Site-Specific Gene Expressions

A, Different sites from which keloid fibroblasts were isolated from the shoulder lesion for comparison of MMP gene expressions among the different sites: i, superficial side; ii, superficial center; iii, deep center; and iv, keratinocytes. Keratinocytes were also isolated from the shoulder lesion. B, Reverse transcription–polymerase chain reaction of MMP gene expressions from primary fibroblasts isolated from different sites of a keloid lesion and compared with the corresponding keratinocytes that lined the epidermis of the lesion, a mixed fibroblast population from the ear, and normal fibroblasts isolated from an eyebrow lift. Fibroblasts from regions closest to the margins of the original wound and the excision site (ie, superficial side and deep center) had higher expressions of MMP1, MMP2, MMP3, and MMP9 compared with those farthest away from the wound boundaries (superficial center). Keratinocytes also expressed the same 4 MMP genes, although at lower intensities, when compared with the superficial side and deep center. The mixed keloid fibroblast had high expressions of MMP genes, whereas normal fibroblasts demonstrated little to no expression of the enzymes.

Graphic Jump Location

Tables

References

Correspondence

CME
Meets CME requirements for:
Browse CME for all U.S. States
Accreditation Information
The American Medical Association is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The AMA designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per course. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Physicians who complete the CME course and score at least 80% correct on the quiz are eligible for AMA PRA Category 1 CreditTM.
Note: You must get at least of the answers correct to pass this quiz.
You have not filled in all the answers to complete this quiz
The following questions were not answered:
Sorry, you have unsuccessfully completed this CME quiz with a score of
The following questions were not answered correctly:
Commitment to Change (optional):
Indicate what change(s) you will implement in your practice, if any, based on this CME course.
Your quiz results:
The filled radio buttons indicate your responses. The preferred responses are highlighted
For CME Course: A Proposed Model for Initial Assessment and Management of Acute Heart Failure Syndromes
Indicate what changes(s) you will implement in your practice, if any, based on this CME course.
NOTE:
Citing articles are presented as examples only. In non-demo SCM6 implementation, integration with CrossRef’s "Cited By" API will populate this tab (http://www.crossref.org/citedby.html).
Submit a Comment

Multimedia

Some tools below are only available to our subscribers or users with an online account.

Sign In to Access Full Content

Related Content

Customize your page view by dragging & repositioning the boxes below.

Articles Related By Topic
Related Topics
PubMed Articles
Jobs
brightcove.createExperiences();