Anti-Aging β-Klotho Gene-Activated Scaffold Promotes Rejuvenative Wound Healing Response in Human Adipose-Derived Stem Cells

Keywords: anti-aging; β-Klotho; gene-activated scaffold; adipose-derived stem cells; angiogenesis; matrix deposition; rejuvenative healing

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1. Introduction 

Wound healing is a complex biological process that requires a tight orchestration of multiple cellular events [1]. However, in the aging population, cellular events are disrupted, leading to delayed healing [2]. The disruption in the healing mechanisms arises partly due to the reduced homing of the progenitor cells from the bone marrow and other prevailing metabolic diseases such as diabetes [3,4]. In wound management, the application of biomaterial scaffolds is becoming more common as a treatment. Biomaterial scaffolds protect the wound from infection, absorb wound exudates, and keep the wound moist to prevent tissue necrosis [5]. Therapeutics can also be loaded into biomaterial scaffolds and promote faster healing in hard-to-heal wounds [1]. However, biomaterial scaffolds alone may not effectively orchestrate the multiple signaling cascades occurring within the wound. Delivering stem cells is sometimes proposed as a solution for moderating the complex signaling events in the wound [6]. 

Stem cells secrete paracrine factors and can differentiate into cells of multiple tissue lineages [7]. Stem cells are typically delivered into the wound through intradermal injections [6], topical spraying [8], or as tissue-engineered grafts [9]. In recent years, the application of tissue-engineered grafts for chronic wound healing has gradually gained wider acceptance because of their ability to heal wounds rapidly [9–11]. Apligraf® and Dermagraft® are two of the widely used Food and Drug Administration (FDA)-approved bioengineered constructs for chronic wound treatment [12,13]. However, the generation of these grafts requires prolonged cell culture to produce high cell numbers [14]. When using stem cells, prolonged culture can increase cellular senescence and diminish the stemness of the stem cells [15,16]. Therefore, maintaining the stemness of the stem cells is central to achieving the optimal therapeutic response. Therapeutic gene delivery to the stem cells using non-viral vectors is a potential strategy to enhance stem cells’ functionality [17]. Traditionally, the cells are transfected in 2 D cultures and later transplanted in vivo [18]. However, platforms such as the gene-activated scaffolds, consisting of biomaterial scaffolds functionalized with nanoparticles carrying the therapeutic transgene [19], that our group has been pioneering offer an alternative solution for transfecting the cells within a 3-D environment. This study focused on developing a gene-activated version of the 3 D collagen-chondroitin sulfate (coll-CS) scaffold that has tremendous clinical translation potential, as its composition is similar to that of Integra’s Dermal Regeneration Template (DRT), a clinically approved scaffold for chronic wound healing [20]. We primarily use a cationic polymer called polyethyleneimine (PEI) to condense DNA plasmids encoding for growth factor genes such as the stromal-derived factor-1 alpha and nerve growth factor and assemble them into charged nanoparticles [21–23]. These charged nanoparticles are then soak-loaded into our coll-CS scaffold to generate the gene-activated scaffolds. Our previous studies have also shown that the PEI-based gene-activated scaffold can cause transient overexpression of the therapeutic transgene in a range of wound-healing cells and promote their regenerative abilities [21,24,25]. Traditionally, the gene-activated scaffold was developed to target host cells and promote local repair [26]. However, emerging evidence indicates that cell-laden gene-activated scaffolds bear superior potency to regenerate complex tissue structures than gene-activated scaffolds alone [27]. 

Cellular abundance is one of the crucial requirements for the generation of tissue-engineered grafts [28]. The adipose tissue is a rich source that can supply a large number of stem cells. The adipose tissue contains as many as 500 times the stem cells for the same mass of bone marrow, a traditional tissue source for stem cell extraction [29]. Moreover, the stem cells can be extracted from the adipose tissue using a minimally invasive liposuction process [29]. Our recent study found that human adipose-derived stem cells (ADSCs) demonstrate excellent biocompatibility with the coll-CS scaffold. Using a pro-angiogenic gene-activated scaffold further improved the ADSCs’ regenerative responses [30]. However, the ADSCs tend to lose their stemness upon expansion [31]. Previously, we found that an anti-aging protein β-Klotho could significantly improve the proliferation of healthy and diabetic human ADSCs [32]. Most studies generally use the primary variant α-Klotho [33–35] and have shown that the protein can increase the lifespan of ADSCs by activating the telomerase transcriptional activity [33]. α-Klotho overexpressing MSCs have also been found to exert strong anti-fibrotic effects in kidney injury through the inhibition of Wnt/β-catenin signaling [36]. However, the role of β-Klotho in stem cells remains relatively unexplored.

Besides the difference in the research focus between the α- and β-Klotho, the anti-aging Klotho proteins have not yet been fully incorporated into the tissue engineering strategies for wound healing. Therefore, in this study, we sought to study the functional impact of a β-Klotho gene-activated scaffold on human ADSCs for wound healing applications. We first investigated the transcriptional activities of the classical pluripotency factors (Oct-4, Nanog, and Sox-2), proliferation marker (Ki-67), and wound healing regulators (TGF-β3 and TGF-β1) in the ADSCs. We then assessed the bioactivity of the conditioned media generated by the ADSCs-laden gene-activated scaffold to evaluate its paracrine potential. Ultimately, we examined the regeneration of the basement membrane (fibronectin, laminin, and collagen IV) and scar-associated proteins (α-SMA and elastin) in the ADSCs-laden gene-activated scaffold to evaluate controlled tissue maturation.

2. Results 

2.1. β-Klotho Gene-Activated Scaffold Transiently Enhances Human ADSCs’ Stemness and Pro-Reparative Genes

Gene expression analysis first showed that the ADSCs on the gene-activated scaffold overexpressed the β-Klotho gene over 172-fold (p < 0.01) than the gene-free scaffold group on day 3 (Figure 1A). This finding confirmed the efficient interaction of the ADSCs with the gene-activated scaffold. Analysis of the proliferation marker Ki-67 further confirmed that the ADSCs on the gene-activated scaffold also maintained a robust proliferative capacity over 14 days (Figure 1A). Having confirmed the overexpression of the anti-aging β-Klotho gene, we then assessed stem cells’ rejuvenation through the activation of “pluripotency” factors in the ADSCs. Overall, we noted a transient regulation of all three transcription factors over the 14 days (Figure 1B). However, the ADSCs only showed a signifificant increase in the expression of the Oct-4 gene that was sustained until day 14. Specifically, the ADSCs showed a 2.5-fold (p < 0.05) increase in the expression of the Oct-4 gene on day 3 before subsiding to 1.6-fold (p < 0.05) on day 14 relative to the gene-free scaffold group. The next set of genes we investigated were the transforming growth factors beta 1 and 3, which are crucial for regulating scarless wound healing (Figure 1C). The levels of the pro-fibrotic TGF-β1 gene in the β-Klotho overexpressing ADSCs were found to be in par with that of the gene-free scaffold group over the 14 days. However, the levels of anti-fibrotic TGF-β3 were signifificantly (3.6-fold, p < 0.05) elevated during the early days (day 3) and, as anticipated, subsided by about 0.4-fold by day 14, showing a controlled expression of the regulatory gene. 

2.2. β-Klotho Gene-Activated Scaffold

Enhances the Paracrine Potency of Human ADSCs 2.2.1. Pro-Angiogenic Bioactivity The bioactivity test of the CM generated by ADSCs-laden gene-activated scaffold first showed that it could temporally control the metabolic activity in HUVECs. Day 3 CM signifificantly enhanced the metabolic activity in HUVECs, while the use of aged CM from day 14 signifificantly reduced the HUVEC’s metabolic activity (Figure 2A). Having observed this, we then tested the pro-angiogenic impact of the CM from day 3 and 14 on the HUVECs seeded on Matrigel. As anticipated, day 3 CM from the gene-activated scaffold group signifificantly (p < 0.05) enhanced HUVEC tubule formation capacity and their branching (Figure 2B). However, no further rise in the angiogenic activity occurred with day 14 CM compared to that of day 3 CM. Also, the day 14 CM from both the groups exhibited similar pro-angiogenic efficiency (Figure 2C). Collectively, our finding indicates that the gene-activated scaffold transiently expedites ADSCs’ pro-angiogenic response.

pro-fibrotic TGF-β1. ** and * indicates p < 0.01 and p < 0.05 respectively. Data represent mean ± standard deviation (n = 3).

Figure 2. Pro-angiogenic paracrine bioactivity of the ADSCs-laden gene-activated scaffold CM. (A) CM produced by the ADSCs-laden gene-activated scaffold over the 14 days demonstrated an ability to temporally control HUVECs' metabolic activity. (B) Day 3 CM from the gene-activated scaffold group significantly enhanced endothelial tubule formation and its branching compared to that of the gene-free scaffold group.(C) Tubule formation and branching of endothelial cells 6hpost-exposure to CM from the ADSCs seeded gene-activated and gene-free scaffold, * indicates p < 0.05. Data represent mean! standard deviation (n = 3)

2.2.2. Dermal Fibroblast Healing and Maturation

One of the findings from the angiogenesis study is that as the ADSCs-laden gene-activated scaffold matures, it no longer promotes angiogenesis. Furthermore, on day 14their angiogenic potency was similar to that of the gene-free scaffold group (Figure 2B, C)Therefore, we investigated the influence of aged day 14 CM on dermal fibroblast wound closure. Similar to the angiogenesis study result, both groups demonstrated similar levels of fibroblast wound closure with day 14 CM. Specifically, both groups healed the wound by approximately 40% in 12 h (Figure 3A). This finding revealed that during maturation, the gene-activated scaffold group supports but does not promote dermal fibroblast wound closure.

Nevertheless, as the fibroblasts were stimulated with aged CM, we studied if the influences of the expression of matrix proteins involved in wound maturation. As anticipated, dermal fibroblasts stimulated with CM from the gene-free scaffold group abundantly expressed the pro-fibrotic collagen I protein (Figure 3B). On the contrary, fibroblasts stimulated with CM from the gene-activated scaffold group demonstrated 50%lower expression (p < 0.05) of the collagen I protein. Meanwhile, both groups lacked the expression of anti-fibrotic collagen all, and no differences were observed between the two groups. Taken together, it implies that the gene-activated scaffold enhances ADSCswound modulatory response during maturation.

Figure 3. Paracrine influence on dermal healing and maturation. (A) Aged day 14 CM from both the groups exerted similar levels of wound closure activity in human adult dermal fibroblasts by approximately 40% in 12 h.(B) CM from the gene-activated scaffold significantly reduced the expression of pro-fibrotic collagen lin the fibroblasts. " indicates p < 0.05Data represents mean = standard deviation (n = 3). In Figure 3B, GAS and GFS stand for gene-activated scaffold and gene-free scaffold respectively

2.3. B-Klotho Gene-Activated Scaffold Enhances Basement Membrane Regeneration with Improved anti-Fibrotic Response in Human ADSCs

Having observed the gene-activated scaffold's controlled stimulation of ADSCs' functionality, we ultimately investigated the ADSCs' response toward the regeneration of dermal matrices and proteins. The gene-activated scaffold robustly enhanced the regeneration of ADSCs' basement membrane. Specifically, the gene-activated scaffold significantly promoted the deposition of basement membrane components laminin and collagen IVFigure 4A). After the semi-quantitative analysis, we also noted that the matrix deposition followed a trend relative to the gene-free scaffold group. The trend as observed wasfibronectin (1.0-fold) < laminin (2.1-fold, p < 0.05) < collagen IV (8.8-fold, p < 0.01).Once the ability to regenerate the basement membrane was determined, we then assessed the expression of proteins crucial for superior qualitative healing, such as reduced scarring. The ADSCs on the gene-activated scaffold demonstrated a significantly (p < 0.05)reduced expression of the scar-associated contractile protein a-SMA. The a-SMA expression in the ADSCs was lower by 50% relative to the gene-free scaffold group (Figure 4B). Lastly having observed the reduced a-SMA expression, we assessed the expression of the elastic matrix protein elastin. The ADSCs on the gene-activated scaffold demonstrated a relatively superior qualitative elastin expression through the deposition of a mature fibrous network of elastin compared to the ADSCs on the gene-free scaffold (Figure 4B).

Figure 4. Deposition of pro-wound healing matrix proteins by the ADSCs in the gene-activated scaffold on day 14.(A) The ADSCs in the gene-activated scaffold demonstrated significant regen. operation of the basement membrane compared to the ADSCs in the gene-free scaffold. The ADSCs predominantly deposited a relatively mature network of collagen IV proteins, followed by laminin and fibronectin.(B) The ADSCs in the gene-activated scaffold also expressed 2-fold lower of the SMA protein compared to the ADSCs in the gene-free scaffold. In conjunction with basement mem. brane regeneration, the ADSCs in the gene-activated scaffold deposited a qualitatively better elastic matrix. All the images were captured through 20x objective using an IX73 Olympus microscope* and * indicates p < 0.01 and p < 0.05 respectively. Scale bar 50um. Data represent mean standard deviation (n = 3).-SMA and fibronectin were double-immunostained.

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