Cutaneous wound healing in aging small mammals: a systematic review.

Cutaneous wound healing in aging small mammals: a systematic review

Dong Joo Kim, BS1, Thomas Mustoe, MD2, and Richard AF Clark, MD3,4 1

School of Medicine, Stony Brook University Division of Plastic and Reconstructive Surgery, Northwestern University Feinberg School of Medicine 3,4 Departments of Dermatology and Biomedical Engineering Stony Brook University

2

Corresponding Author: Richard AF Clark, MD Departments of Dermatology and Biomedical Engineering HSC T-16, 060 Stony Brook University Stony Brook, NY 11794-8165 TEL: 631-444-7519 FAX: 631-444-3844 email: [email protected]

Running Head: Wound healing in aging small mammals

Keywords: aging, acute wounds, systemic review, animal studies

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1111/wrr.12290

This article is protected by copyright. All rights reserved.

Manuscript under review - CONFIDENTIAL

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ABSTRACT As the elderly population grows, so do the clinical and socioeconomic burdens of non-healing cutaneous wounds, the majority of which are seen among persons over 60 years of age. Studies of how aging effects cutaneous wound healing have become a priority. Human studies will always be the gold standard, but studies have ethical and practical hurdles. Choosing an animal model is dictated by costs and animal lifespan that preclude large animal use. Here we review the current literature on how aging effects cutaneous wound healing in small animal models and, when possible, to compare healing across studies. Using a literature search of MEDLINE/PubMed databases, studies were limited to those that utilized full-thickness wounds and compared the wound-healing parameters of wound closure, re-epithelialization, granulation tissue fill, and tensile strength between young and aged cohorts. Overall, wound closure, re-epithelialization, and granulation tissue fill were delayed or decreased with aging across different strains of mice and rats. Aging in mice was associated with lower tensile strength early in the wound healing process, but greater tensile strength later in the wound healing process. Similarly, aging in rats was associated with lower tensile strength early in the wound healing process, but no significant tensile strength difference between young and old rats later in healing wounds. From studies in New Zealand White rabbits, we found that re-epithelialization and granulation tissue fill were delayed or decreased overall with aging. While similarities and differences in key wound healing parameters were noted between different strains and species, the comparability across the studies was highly questionable, highlighted by wide variability in experimental design and reporting. In future studies, standardized experimental design and reporting would help to establish comparable study groups, and advance the overall knowledge base, facilitating the translatability of animal data to the human clinical condition.

2 Wound Repair and Regeneration This article is protected by copyright. All rights reserved.

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INTRODUCTION The elderly are the fastest growing age group worldwide, with the proportion of the world’s population over 60 years of age expected to double from 11% to 22% between the years 2000 and 2050 [1]. As the elderly population continues to grow, so do the clinical and socioeconomic burdens of post-operative wound dehiscence and non-healing cutaneous wounds, the majority of which occur among persons over 60 years of age [2]. While non-healing wounds are primarily associated with age-related comorbidities, such as diabetic ulcers, pressure sores, and venous stasis ulcers, aging itself is considered an independent risk factor for delayed wound healing. The characteristic changes that occur normally in aged skin, such as decreased proliferation of keratinocytes, dermal atrophy, and impaired macrophage function, have implications for failed or delayed cutaneous wound healing [3, 4] . Alterations in the dynamic, yet precise, events that comprise the complex wound healing process ultimately contribute to its delay [5]. Taken together, the study of aging and its effect on cutaneous wound healing is a priority issue. Human studies will always be the gold standard, as no model can replicate clinical human wound healing. However, many of the early studies in humans lacked the proper adjustments for confounders, such as common comorbidities found in the older population (eg, diabetes, vascular disease), nutritional status, immobilization, and site specificity. In fact, elderly patients can undergo major surgical procedures, such as open-heart surgery [6] and resection of an abdominal aortic aneurysm [7], and most post-operative wounds will heal well as long as comorbidities and other cofounders are not present to adversely affect wound healing. This has led some investigators to question the dogma that cutaneous wound healing is impaired as a function of age [7-10]. Whether differences in wound healing are due to chronological aging, or are principally reflections of a greater reserve capacity in the young population [11] is beyond the scope of this review. Aside from the flaws in experimental design of early studies, experiments with human subjects are often impractical. First, recruiting participants with identical or similar wounds and comparable health statuses for randomized trials is challenging. Second, multiple skin biopsies are often required throughout the course of the experiment for objective measurements of wound healing, presenting a burden to participants [12] that may limit the opportunities to carry out controlled experimentation. Furthermore, ethical considerations may also prohibit the use of human subjects, especially those with impaired wound healing ability.



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In vitro models have been used to map wound repair pathways in fibroblasts and keratinocytes, the predominant cell populations in mammalian skin. However, these models lack key in vivo cues (ie, endocrine and paracrine signaling), which limit their translational applicability [8]. Furthermore, in vivo experimentation is ultimately needed to substantiate in vitro wound healing findings [13]. In selecting an animal model, it is important to consider the strengths and weaknesses of each model in the context of: 1) suitability and relevance, and 2) feasibility. Suitability and relevance determinations are needed as to whether the animal model reflects anatomical, physiological, and biological processes occurring in elderly humans during wound healing [14, 15]; the ideal model being one that reproduces the human clinical condition in its entirety. Feasibility determinations are made as to the practical aspects of experimentation, including availability, procurement, housing and maintenance costs, and lifespan. In addition, investigator familiarity and availability of established reagents, protocols, and body of scientific literature are also important feasibility considerations. Small mammal models of aging and wound healing, such as mice, rat, and rabbit models, generally have lower suitability and relevance, but higher feasibility than do large mammal models of aging. The skin anatomy and physiological characteristics of these smaller mammals is quite different from human skin [8, 16]. For instance, rats and mice possess a subcutaneous panniculus carnosus muscle that is absent in humans. This muscle contributes to cutaneous wound healing by both contraction and collagen formation [13, 15]. Furthermore, rats and mice are described as “loose-skinned” animals due to their skin’s redundancy and elasticity, as well as its lack of adherence to underlying structures. The properties of “loose” skin allow wound contraction to play a significant role in wound healing [16, 17]. Wound contraction is usually more rapid than epithelialization, and accelerates the overall healing time of rats and mice. Small mammals also have a greater density of hair follicles, which are involved in epithelialization, compared to human skin. Such differences make small mammal wound outcomes difficult to compare to humans, and brings to question the translational ability of small mammal-derived data to the human clinical condition. Small mammal models have been used to study the effects of common comorbidities that are a major cause of delayed healing in the elderly, such as diabetes [18], ischemia and peripheral arterial disease [19-21], and decline of sex steroid hormones [22, 23], often demonstrating delayed healing that is synergistic with chronological aging. However, it remains unclear whether aged animals have naturally occurring comorbidities


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that impact their ability to heal. Furthermore, these conditions are not routinely screened for in current animal models of wound healing in which the emphasis is not on these comorbidities. This poses the question of whether the findings of delayed wound healing are the function of chronological aging or unidentified confounders. In addition, systematic comparison of the genomic response between human inflammatory diseases and murine models has shown poor correlation, and challenges the notion that results from animal research can mimic human disease [24]. Nonetheless, the advantage of using small mammal models is in large part due to their high feasibility. Smaller research animals are typically less expensive, cost less to maintain, and are easier to handle. Mice and rats are readily accessible, including thousands of mutation, knockout, and transgenic strains available to study disease pathophysiology [8, 25]. There are also a wide variety of reagents specific to mice and rats available for research purposes. As such, there is often more investigator familiarity with these smaller mammals. Furthermore, these smaller mammals have shorter gestation times and shorter lifespans, making the study of chronological aging more practical. The wound healing process is also accelerated in these animals, such that it is possible to study the process over days rather than weeks, as is the case for larger mammals and humans [17]. Together, these feasibility advantages have led to a larger scientific knowledge base from these animals. On the other hand, large mammal models of aging and wound healing, such as porcine models, generally have higher suitability and relevance, but lower feasibility. Anatomically and physiologically, porcine skin is quite similar to human skin [12, 26]. Pigs, like humans, have sparse body hair, which has implications for re-epithelialization of wounded skin, i.e. the lower density of body hair in pigs and humans means reepithelialization occurs mainly through interfollicular stem cells. Pigs, like humans, lack a panniculus carnonsus muscle, and thus do not rely on muscle contraction for wound healing. Pigs are also tight-skinned animals and, like humans, close wounds to a significant extent through re-epithelialization [12]. The biological similarities in wound healing between pigs and humans are undeniable. However, the study of aging and wound healing is limited in pigs by its low feasibility. In addition to expensive costs, the lifespan of pigs is chronologically matched to humans, such that the test animals could outlive their researchers, and thus would take too much time to obtain useful results. Therefore, the literature of aging and wound healing has primarily utilized mouse, rat, and rabbit models, and these models will be the focus of this review.



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The purpose of this paper is to review the current literature on how aging effects cutaneous wound healing in mouse, rat, and rabbit models. Wound healing is usually assessed by one or more of the following measures: 1) rate of or time to wound closure, 2) some factor of dermal healing (eg, granulation tissue fill, protein accumulation), and/or 3) tensile strength [10]. For this review, studies were selected in which wounds were inflicted in cohorts of young and aged animals to compare the effects of true, chronological aging on the wound healing parameters of wound closure, re-epithelialization, granulation tissue fill, and tensile strength. From the comparison of these parameters between the different animal models, we will attempt to determine the current knowledge on aging and wound healing, and to potentially build a consensus for a standard animal model to be used in future studies of aging and wound healing.

MATERIALS AND METHODS Review search strategy A literature search using MEDLINE/PubMed databases for the terms aging, wound, and healing identified 1500 articles. Replacing the term aging with ageing identified an additional 94 articles for a total of 1594 articles. For the purpose of this review, we were interested in mouse, rat, and rabbit models of aging and cutaneous wound healing in which full-thickness, incisional or excisional, wounds were inflicted onto animals, with the measured wound healing parameters of wound closure, re-epithelialization rate, granulation tissue fill, and/or tensile strength. In an attempt narrow our search to our parameters of interest, we applied different combinations of the terms aging/ageing + healing/wound healing/wound-healing + skin + animal/mouse/mice/murine/rat/rodent/rabbit, which generated 276 studies all confined to the 1594 articles. However, this approach resulted in the exclusion of a number of important studies that met our inclusion criteria. Therefore, the 1594 articles from the original search were designated as the initial pre-screened pool of articles from which this literature review was derived. After manually screening the unfiltered pool of articles, we found that 107 articles met our inclusion criteria for aging and cutaneous wound healing. Forty-one of these articles involved studies of non-chronological aging; for instance, models that compared young mice to their ovariectomized/castrated young counterparts to simulate the physiologic decrease in estrogens/testosterone that occurs naturally with aging. This neglects the overall age-related changes that occur beyond hormonal


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changes and therefore were not used in this analysis. Sixty-six articles involved true aging in which young animals and their chronologically aged counterparts were studied. Every effort was made to include only prospective, randomized, controlled studies with well-defined, objective outcome measurements. The 66 studies of true aging and cutaneous wound healing are outlined in this review.

Wound closure analysis The experimental designs of the selected studies varied widely. The wound types ranged from 3 mm punch biopsies to 5 mm excisional wounds to 4 cm incisions, resulting in different baseline wound areas (day 0 data). In many cases, the baseline wound area was not reported altogether, with wound area measurements beginning at a later time-point. This had important consequences for wound closure determinations, since wound area at subsequent time-points could not be compared to a baseline wound area to determine wound closure as a percentage of baseline. Therefore, the wound area that remained on each day of wound examination was considered. In addition to the differences in wound area at baseline, the days on which the wounds were examined also varied, with some studies tracking wound area daily until closure and others examining wound area at set intervals. This made comparisons of wound closure data across different studies difficult. To adjust for these considerations, the ‘Wound Area Ratio’ of old to young animals was calculated for statistically significant time-points when possible and averaged together for each study, where:

An average Wound Area Ratio (Old:Young) of 2 would indicate that at any given time-point, the remaining wound area in an old animal is twice as large as the same wound in a young animal. The average Wound Area Ratios (Old:Young) could then be compared across different studies. Fold differences in Wound Closure Ratio between young and old animals was preferred, but could not be standardized across the studies due to the absence of reported baseline wound areas in many studies. Rate of Closure (%/day) was calculated from linear regression analysis of wound areas at their respective time points. This was not possible for studies without a recorded baseline wound area (day 0 data). Time at 50% Wound Closure (days) was determined based on graphing wound area versus time. Time at 50%



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Wound Closure (days) has been suggested by some groups as an ideal index of wound closure because of the variability that exists with respect to initial and later healing rates. Time at 100% Wound Closure (days) was determined based on graphing wound area versus time. This was also problematic, as most studies did not track wound area until complete closure. In those studies, Time at 100% Wound Closure was estimated based on the calculated Rate of Closure. In the few cases where wounds areas were tracked to full closure, the majority examined the wounds at set intervals (ie, day 3, 7, 14). In such cases, Time at 100% Wound Closure data had the possibility of being inaccurate because without daily wound area examinations, it could not be known whether 100% wound closure was achieved at an earlier date than reported. Re-epithelialization analysis The experimental designs of the selected studies varied widely, from 1 cm full-thickness incisions to 6 mm punch biopsies in the Ear Wound Model. Similar to the Wound Area Ratio (Old:Young) used in Wound Closure analysis, the Re-epithelialization Ratio (Old:Young) was calculated for each statistically significant time-point and averaged to compare re-epithelialization across different studies. Time at 50% Reepithelialization (days) and Time at 100% Re-epithelialization (days) were calculated as above.

Granulation tissue fill analysis Comparison of granulation tissue fill across different studies was challenging because each individual study examined different aspects of granulation tissue fill (ie, hydroxyproline content, fibroblast populations, etc) and used a variety of methods. Data were presented as a summary of key findings from each study. Tensile Strength Analysis The experimental designs of the selected studies varied widely, from 4 cm to 3 inch incisions, and timepoints of tensile strength assessments ranging from day 4 to week 13. Similar to the Wound Area Ratio (Old:Young) and Re-epithelialization Ratio (Old:Young), the Tensile Strength Ratio (Old:Young) was calculated for each statistically significant time-point and averaged to compare tensile strength across different studies.


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RESULTS Wound closure From our pool of 66 articles, 24 examined wound area closure in incisional and excisional wounds—18 studies in different mouse strains [27-43] (Table 1) and 6 studies in different rat strains [21, 44-48] (Table 2). Based on Wound Area Ratios of old to young animals (O:Y), the majority of selected studies in mice and in rats found a significantly higher area of unclosed wounds in old animals versus young animals for any given timepoint, indicative of delayed wound closure as a function of aging. O:Y Wound Area Ratio could not be calculated for Boulter et al’s study because a different scale was used to assess wound area between young and old mice, and conversions were not available to make the determinations. In Reed et al, there was no statistical difference in wound closure despite an O:Y Wound Area Ratio of 1.47 because there was no statistical difference in the reported wound area between young (0.44±0.14 mm2) and old (0.65±0.24 mm2) mice at the only time-point (day 11). In Tyner et al, the O:Y Wound Area Ratio was 1.09 with no statistical significance, while in Moor et al, the O:Y Wound Area Ratio was 1.06 with no statistical difference in wound area between young and aged mice on days 2, 5, 7, 10 and 14 post-wounding. The calculated Rate of Closure (%/day) was lower for older animals in the majority of selected mouse studies and in all selected rat studies, supportive of delayed wound closure with aging. The Rate of Closure could not be calculated in studies that did not report a baseline (day 0) wound area. The Time at 50% Closure was delayed for the older cohorts in the majority of mouse and rat studies. Time at 100% Wound Closure was challenging to analyze because the majority of studies did not track wound closure to completion. Among those that did, the majority of studies analyzed wound area at set intervals (ie, 3, 7, 14 days), with 100% wound closure usually reported on the final day of wound analysis. Unless wound area is assessed daily, the exact time at 100% wound closure cannot be accurately reported.



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O:Y Wound Area Ratio was averaged for different strains of each species (Table 3). For mice, the Balb/c (1.42), Hairless JapanSLC (1.63), C57L/lcrfat (4.1), and C57BL/6J (1.40) strains showed a significantly higher average O:Y Wound Area Ratio, indicative of delayed wound closure as a function of aging in those strains. The Hairless SKH-1 strain demonstrated an increase in O:Y Wound Area Ratio of 1.09 of no statistical significance. However, the Peromyscus strain showed a significantly lower average O:Y Wound Area Ratio of 0.52, indicative of accelerated wound closure as a function of aging in that strain. This finding was surprising to the authors, Cohen et al, who speculated that as a result of selection from a randomly outbred colony, the aged Peromyscus mice could represent a “highly selected group of survivors with a greater capacity than younger mice for efficient wound repair”, indicative of different gene pools. For rats, the Fisher 344/Brown Norway (2.04), Wistar (2.04), and R. rattus (1.67) strains showed a significantly higher average O:Y Wound Area Ratio, indicative of delayed wound closure as a function of aging in those strains. The Fisher344 strain demonstrated an increase in O:Y Wound Area Ratio of 1.06 that was not statistically significant. Comparing mice to rats, the average O:Y Wound Area Ratio across the selected mice studies was 1.46, which was less than the 1.89 for the selected rat studies. Furthermore, both young and old mice had a higher Rate of Closure, and earlier Time at 50% Closure and Time at 100% Closure, indicative of faster wound closure than their rat counterparts.

Re-epithelialization From our pool of 66 articles, 9 examined re-epithelialization in incisional and excisional wounds—4 studies in different mouse strains [27, 41, 49, 50], 2 studies in different rat strains [48, 51], and 3 studies in New Zealand White rabbits [20, 52, 53] (Table 4). Based on the O:Y Re-epithelialization Ratio of old to young animals, 8 of the 9 studies found a significantly lower wound re-epithelialization in old animals versus young animals for a given time-point, indicative of delayed re-epithelialization with aging. In Bonomo et al’s study of New Zealand White rabbits, complete re-epithelialization had already taken place at the study’s single timepoint (Day 26), and thus, the calculated O:Y Re-epithelialization Ratio was 1. Of note, most of the studies only had statistically significant data from a single time-point. Therefore, the rate of re-epithelialization was not summarized in Table 4. For Mogford et al, re-epithelialization data was presented for days 4, 5, 6, and 7 for


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young rats, for which a rate of re-epithelialization of 19.9 %/day was calculated based on the linear regression analysis. In Adams et al, the Time at 50% Re-epithelialization was delayed for the old mice compared to the young mice (4 days versus 2.73 days, respectively). In Mogford et al, the Time at 50% Re-epithelialization was reported to be delayed for the old rats compared to the young rats (7 days versus 4.7 days, respectively). Time at 50% Re-epithelialization could not be calculated for Ashcroft et al, Wu et al, and Xia et al’s studies because these studies only reported re-epithelialization data for a single time-point. In Adams et al, the Time at 100% Re-epithelialization was 14 days for both young and old mice. However, re-epithelialization was assessed on days 3, 7 and 14, and it is unknown whether full re-epithelialization occurred at an earlier time than reported. In Ashcroft et al, all wounds had re-epithelialized regardless of age by day 7. However, re-epithelialization was not monitored daily. In Mogford et al, a Time at 100% re-epithelialization of 5.03 days was calculated for young rats based on the rate of re-epithelialization. It could not be determined for old rats because only day 7 data was available. In Wu et al and Xia et al, only one time-point was available and Time at 100% reepithelialization was not tracked. Re-epithelialization data was averaged for different strains of mice and rats, and for the three studies in New Zealand White rabbits (Table 5). Bonomo et al’s study was not included when calculating the average Re-epithelialization Ratio (O:Y) for New Zealand White rabbits because complete reepithelialization had occurred at time of measurement.

Granulation Tissue Fill From our pool of 66 articles, 26 examined granulation tissue fill by various means in incisional and excisional wounds—9 studies in different mice strains [18, 36, 37, 41, 49, 50, 54-56] (Table 6), 12 studies in different rat strains [44, 48, 51, 57-65] (Table 7), and 5 studies in New Zealand White rabbits [20, 52, 53, 66, 67] (Table 8). Each study examined its own set of granulation tissue fill measures, ranging from hydroxyproline content to angiogenesis, and each study used its own methodology, typically based on histologic analysis. Thus, comparison of granulation tissue fill across the different animal models was not possible. Granulation tissue fill was decreased or delayed overall in aged mice compared to young mice. Ashcroft et al demonstrated delayed ECM matrix deposition and decreased angiogenesis in aged C57BL/lcrfa mice compared to their young counterparts. Studies in C57BL/6J mice demonstrated decreased granulation tissue volume and wound



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fibroblasts in aged C57BL/6J mice compared to their young counterparts. Loh et al demonstrated significantly decreased wound capillary density in aged C57BL/6J mice compared to young mice, while Reed et al showed that angiogenesis was similar in the dermis of young and aged C57BL/6J mice at later stages of tissue repair. Reed et al explained that after initial age-related deficits, the angiogenic response in aged tissues approaches that of young tissues in later stages of repair. In Balb/c mice, studies by Swift et al demonstrated delayed wound angiogenesis and collagen deposition in aged mice compared to young mice. Granulation tissue fill was decreased or delayed overall in aged rats compared to young rats. Ballas et al demonstrated decreased myofibroblasts and decreased overall granulation tissue development in old Fisher 344/Brown Norway rats compared to their young counterparts on Days 6 and 10, with normalization by Day 14. Studies in Fisher 344 rats demonstrated decreased hydroxyproline levels, fewer collagen bundles, and reduced overall granulation tissue formation in wounds in old rats compared to their young counterparts. Studies in Wistar rats demonstrated decreased hydroxyproline levels, collagen fibers, and fibroblasts in old rats compared to young rats. Kanta et al demonstrated increased weight of granulation tissue formed in aged Wistar rats compared to young Wistar rats, but proposed no explanation for the finding. Studies in Sprague-Dawley rats demonstrated delayed granulation tissue fill by various measures. Gupta et al demonstrated a reduction in the activity profiles of glycolytic enzymes associated with energy metabolism in wounds in old rats compared to their young counterparts. The authors explained that wound healing processes such as neovascularization and granulation tissue formation require enhanced energy utilization, with anaerobic glycolysis serving as the main energy pathway in the skin. Furthermore, the authors proposed that the trend towards reduced activities of these glycolytic enzymes may attribute to impaired wound healing in aged rats. Sobin et al demonstrated that the wound microvasculature in aged rats begins as Periodic Acid-Schiff (PAS) negative or “young” microvasculature but becomes strongly PAS positive or “old” during the ensuing weeks. However, wound microvasculature in young rats remained PAS negative or “young” throughout the 2 month observational period. Holm-Pedersen and Zederfeldt demonstrated increased mean thickness of wounded skin in older rats compared to young rats. Granulation tissue fill was decreased or delayed overall in aged New Zealand rabbits compared to young New Zealand rabbits. Wu et al demonstrated decreased granulation tissue formation in aged rabbits compared to young rabbits, with a calculated granulation tissue ratio of old rabbits to young


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rabbits (O:Y) of 0.13, while Xia et all demonstrated an O:Y granulation tissue ratio of 0.52. Brucker et al demonstrated a significant decrease in the gene expression of PDGF-Rβ, a central mediator of the wound repair response, in wounds of old rabbits versus young rabbits. In Bonomo et al’s study, complete wound healing had occurred by harvest on Day 26, at which time there was a non-significant decrease in new granulation tissue formation and new collagen deposition in old rabbits compared to young rabbits.

Tensile Strength From our pool of 66 articles, 15 examined tensile strength in incisional wounds—4 studies in different mouse strains [18, 68-70], 10 studies in different rat strains [19, 44, 57, 64, 71-76], and 1 study in New Zealand Rabbits [66] (Table 9). In C57BL/6 mice, there was a range in O:Y Tensile Strength Ratio of old to young mice of 0.32 (day 7) to 1.93 (week 6). It is possible that early in wound healing, tensile strength was lower in old C57BL/6 mice compared to young C57BL/6 mice, while late in wound healing, tensile strength was higher in old C57BL/6 mice compared to young C57BL/6. Ershler et al demonstrated increased fibrous proliferation upon histologic examination of samples from old mice, and proposed that local fibrous containment was more prominent in the older host. They proposed that the mechanism for the more vigorous fibrotic response in older mice may be related to local factors, such as epidermal growth factors and capacity for angiogenesis, but did not elaborate further. Ballas et al demonstrated a significantly lower tensile strength in old Fisher 344/Brown Norway rats on days 10 and 14 for an average O:Y Tensile Strength Ratio of 0.68, while Beck et al showed a significantly lower tensile strength in old Fisher 344 rats on day 7 for an average O:Y Tensile Strength Ratio of 0.77. For Wistar rats, there was a range in O:Y Tensile Strength Ratio of old to young rats of 0.33 (day 4) to 1.06 (day 20). Petersen et al and Seyer-Hansen et al examined tensile strength properties of 4 cm, midline abdominal incisions. Petersen et al demonstrated a significantly lower tensile strength in old rats compared to young rats on day 4 for a Tensile Strength Ratio (O:Y) of 0.33, while Seyer-Hansen et al found no significant difference in tensile strength in old rats compared to young rats on day 7 for a Tensile Strength Ratio (O:Y) of 1.04. The other studies in Wistar rats involved dorsal incisions, which showed no significant difference in tensile strength between young and old Wistar rats at 1, 3, 8, and 13 weeks. It is possible that tensile strength is lower in old Wistar rats compared to young Wistar rats early in the wound healing process (ie, Day 4), but



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the difference becomes insignificant later in the wound healing process (ie, Day 7 and beyond). However, Cox et al demonstrated an average O:Y Tensile Strength Ratio of 0.73 at day 14, 21, and 28 time-points, indicating lower tensile strength in old Wistar rats compared to young Wistar rats at these later time-points. HolmPedersen and Zederfeldt demonstrated a significantly lower tensile strength in old Sprague-Dawley rats compared to young Sprague-Dawley rats on days 4, 7, 14, and 21 post-wounding, for an average O:Y Tensile Strength Ratio of 0.60. Thicker skin of older rats may account for the diminishing differences in tensile strength in old versus young rats at later time points observed in some studies. In New Zealand White rabbits, Wu et al demonstrated a non-significant decrease in tensile strength in old rabbits compared to young rabbits on day 14, with a O:Y Tensile Strength Ratio of 0.87.

DISCUSSION The purpose of this review was to outline the current state of literature on the effects of aging on cutaneous wound healing in mouse, rat, and rabbit models, with the hope that the information gathered would help investigators to compare across studies to select an animal model and experimental approach moving forward. The current approach is such that each study group has its own agenda and isolates specific events and mechanisms of a partitioned wound healing process, ranging widely from macrophage infiltration during the inflammatory phase of healing to the effects of androgens on collagen deposition. In this review, we limited our scope to studies that utilized the full-thickness model, which is commonly used among mouse, rat, and rabbit experiments to excise the underlying panniculus carnosus muscle in attempts to increase translatability to the human condition. In the full-thickness model, wound healing is often monitored on the basis of wound closure, extent of re-epithelialization, granulation tissue fill, and tensile strength [16]—the key parameters to which our review was confined. From our review of studies in mice, we found that wound closure, re-epithelialization, and granulation tissue fill were delayed or decreased with aging when comparing studies across almost all different mouse strains. The Peromyscus strain was different from the other stains in that older mice healed faster than younger mice. Clearly this study needs to be confirmed and if so done, should be expanded to elucidate the


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underlying mechanistic cause of this extraordinary result. In general, aging mice tend to show lower tensile strength early in the wound healing process, but greater tensile strength later in the healing process. Similarly, from our review of studies in rats, we found that wound closure, re-epithelialization, and granulation tissue fill were delayed or decreased with aging when comparing studies across different rat strains overall. Aging in rats may be associated with lower tensile strength early in the wound healing process, but no significant in tensile strength different between young and old rats later in the wound healing process. The finding of lower tensile strength early in the healing process may be related to the fact that ischemia affects wound healing more profoundly in older rats than younger ones, with ischemia during early wound healing (within the initial 2-3 week critical period) proposed to have the most profound effect on healing [74]. However, a thicker dermis in old versus young rats is a confounding factor in these comparisons. From our review of studies in New Zealand White rabbits, we found that re-epithelialization and granulation tissue fill were delayed or decreased overall with aging, while a single study by Wu et al demonstrated no significant difference in tensile strength with aging. Comparing mice and rats by species, it appeared that mice, both young and old, had a relatively higher rate of wound closure compared to their rat counterparts. An important difference in aging between rats and mice versus rabbits is the relatively longer life span of rabbits (8-10 years when kept as pets versus 2 years in mice and rats). Furthermore, mice and rats lack superoxide dismutase, which has been proposed to increase their susceptibility to oxidative stress than longer-lived animals; however, mice and rats constitutively express telomerase while rabbits, like humans, do not [77]. Nevertheless, a prevailing theme that arose while attempting to make comparisons across these animal models of different strains and different species was the need for standardization of wound type, size, and assessments including time-points of assessment. While similarities and differences in key wound healing parameters were noted between different strains and species, the comparability across the studies was highly questionable. The issues with cross-study comparability were in large part due to the wide variability in the experimental design of studies and, to a lesser extent, the insufficient reporting or explanation of key details. This is best illustrated by the studies that investigated wound closure, the wound healing parameter that is perhaps most associated with a meaningful clinical outcome. Wound types ranged from 1 cm incisions of unspecified wound areas to 6 mm diameter



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excisions made with scissors to 3.5 mm punch biopsies, resulting in a variety of baseline (day 0) wound sizes and shapes. These are important considerations in wound healing studies as they have implications for 1) elastic forces of the surrounding skin and wound contraction [78], and 2) the physical and metabolic stress of a large wound relative to total body surface area, which will independently influence wound healing [15, 17]. In some studies, baseline wound areas were not reported, while in other studies, baseline wound areas were implicit in the wound type (ie, a 4 mm punch biopsy for an expected area of 12.6 mm2). However, it is important to acknowledge that the baseline wound area may not always reflect the original intended area. Without a reliable baseline wound area, the rate of wound closure could not be determined for some of the studies, losing a convenient wound closure measure that in other circumstances could be used to compare across studies. Time at 100% Wound Closure, which is considered the gold standard for wound healing in human studies, could not be determined in the majority of the studies because investigators opted to use partial closure endpoints. In those studies that tracked wound closure to completion, the majority of studies examined wound area at spaced intervals (ie, at 3, 7, 14 days), rather than daily. Unless wound area is assessed daily, the exact time at 100% wound closure cannot be accurately reported. The absence of reliable Time at 100% Would Closure data has implications for overall translatability to the human clinical condition. Furthermore, in studies that examined tensile strength, incisions varied in size, location, and time-points at which they were assessed. When studying tensile strength, it is important to recognize that the placement of the affects wound healing. Tensile strength was found to decrease in dorsal incisions as a function of how caudally they were inflicted, possibly secondary to differences in vascularity at different skin regions [79]. The majority of the studies in this review measured tensile strength at a single time-point, ranging from 4 days to 13 weeks. The comparability of these measurements across studies was limited without additional time-points. Despite some variation, animal models of aging and wound healing in large part share reproducibility. Moving forward, reproducibility should be combined with standardization in experimental design and reports to establish comparability between study groups. Possible standardization in design and reports are suggested as follows. Animal models of healing of full thickness wounds fall into the categories of surgical incisions, (wounds approximated with sutures or surgical clips), and excisional wounds that are left to heal by reepithelization and wound contracture. For incisional wounds, the critical parameter is gain in tensile strength,


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which does not meaningfully begin until the onset of collagen synthesis after day 3. We suggest that tensile strength should be measured at least at day 7, and day 14 time points. Beyond that time period most gains in tensile strength are due to collagen remodeling and collagen cross linking. Intermediate time points of day 5 and day 10 are also useful but less critical and less necessary for standardization. For excisional wounds that heal by secondary intention, the most critical parameter is time to complete reepithelization, a function of species and wound size. In addition, healing rate is also a useful parameter.

Wound size decrease can be

easily calculated from wound diameter with assumption that the wound is circular. Alternatively it can be directly measured. At least two time points prior to closure should be measured, since the healing rate slows near completion of healing. The rate of healing can be best expressed as the rate in the reduction of wound area, with the initial wound size included. In addition, a calculation of the percentage of closure due to wound contraction versus epithelization should also be included based on histological analysis at the time wound closure. Once types of wounds and their assessment are standardized across animal species, stressors, such as ischemia/reperfusion or diabetes, could be added to determine whether such comorbidities would accentuate the differences in healing between young and old animals [20], and potential therapies could be tested to determine whether healing would be promoted in different species, young or old. Not only will this lead to advancement of the overall knowledge base, it will facilitate the translational application of animal data to the human clinical condition.



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