Abstract
Decellularized extracellular matrices (dECM) have strong regenerative potential as tissue engineering scaffolds; however, current clinical options for dECM scaffolds are limited to freeze-drying its native form into sheets. Electrospinning is a versatile scaffold fabrication technique that allows control of macro- and microarchitecture. It remains challenging to electrospin dECM, which has led researchers to either blend it with synthetic materials or use enzymatic digestion to fully solubilize the dECM. Both strategies reduce the innate bioactivity of dECM and limit its regenerative potential. Herein, we developed a new suspension electrospinning method to fabricate a pure dECM fibrous mesh that retains its innate bioactivity. Systematic investigation of suspension parameters was used to identify critical rheological properties required to instill “spinnability,” including homogenization, concentration, and particle size. Homogenization enhanced particle interaction to impart the requisite elastic behavior to withstand electrostatic drawing without breaking. A direct correlation between concentration and viscosity was observed that altered fiber morphology; whereas, particle size had minimal impact on suspension properties and fiber morphology. The versatility of this new method was demonstrated by electrospinning dECM with three common decellularization techniques (Abraham, Badylak, Luo) and tissue sources (intestinal submucosa, heart, skin). Bioactivity retention after electrospinning was confirmed using cell proliferation, angiogenesis, and macrophage polarization assays. Collectively, these findings provide a framework for researchers to electrospin dECM for diverse tissue engineering applications.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 640-656 |
| Number of pages | 17 |
| Journal | Bioactive Materials |
| Volume | 41 |
| DOIs | |
| State | Published - Nov 2024 |
Keywords
- Biological scaffolds
- Electrospinning
- Extracellular matrix
ASJC Scopus subject areas
- Biotechnology
- Biomaterials
- Biomedical Engineering
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In: Bioactive Materials, Vol. 41, 11.2024, p. 640-656.
Research output: Contribution to journal › Article › peer-review
}
TY - JOUR
T1 - Suspension electrospinning of decellularized extracellular matrix
T2 - A new method to preserve bioactivity
AU - Jones, Sarah
AU - VandenHeuvel, Sabrina
AU - Luengo Martinez, Andres
AU - Birur, Ruchi
AU - Burgeson, Eric
AU - Gilbert, Isabelle
AU - Baker, Aaron
AU - Wolf, Matthew
AU - Raghavan, Shreya A.
AU - Rogers, Simon
AU - Cosgriff-Hernandez, Elizabeth
N1 - Tissue engineering scaffolds composed of decellularized extracellular matrices (dECM) have shown a tremendous capacity to support regeneration in numerous applications [1]. The dECM guides tissue remodeling by providing structural and biological cues with its complex composition of fibrous proteins, proteoglycans, growth factors, cytokines, and mRNA [1\u20134]. Cells interact with and resorb the dECM, leading to the release of bioactive products, such as chemoattractive low molecular weight proteins, angiogenic growth factors, and mRNA [1,4,5]. Tissue-specific dECM has successfully promoted angiogenic, myogenic, neurogenic, and immunomodulatory behavior by supporting cell recruitment, proliferation, and tissue-specific differentiation [6\u201312]. Small intestinal submucosa (SIS) is one of the most prominent dECM scaffolds on the market because it contains the high levels of growth factors and nutrients necessary to support the continual regeneration of the intestinal lining. SIS scaffolds have shown success in regenerating airway [13], abdominal wall [14,15], diaphragm [16], intestine [17], bladder [18], rotator cuff [19], skin [20\u201322], and urethra [23] tissue in clinical trials [1].The relationship between the storage and loss modulus of the suspensions across a range of strain amplitudes was characterized (Fig. 2C and D). In all cases, the suspensions acted as viscoelastic solids at small amplitudes, as noted by the dominance of the storage modulus over the loss modulus, and viscoelastic liquids at large amplitudes, where the loss modulus is larger than the storage modulus. Although the transition from primarily elastic behavior to primarily viscous behavior is complex and transient, the yield point can be estimated from the crossover between the storage and loss modulus. Above the crossover strain, the suspension flows unrecoverably, while below the crossover strain, the suspension undergoes more elastic deformation that can be recovered after the strain is removed. Homogenization of the suspensions had a direct effect on the crossover point. The suspensions that were homogenized had a larger crossover strain amplitude with the homogenization of the SIS sheet having a greater impact than the homogenization of the suspension. The amplitude sweep was conducted in both ascending and descending fashion to determine if the rheological behavior of the suspensions is time-dependent or thixotropic. The suspensions that were homogenized exhibited a larger hysteresis loop indicating increased thixotropic behavior. Similar responses have previously been observed in hydrogels [70]. The homogenization of the SIS sheet had a greater impact on thixotropy than the homogenization of the suspension. Lastly, a flow ramp was conducted to evaluate the rate-dependence of the viscosity of the suspensions (Fig. 2E). Although viscosity alone did not fully predict the spinnability of suspensions, it is an important parameter to assess the full rheological profile of the suspensions. At shear rates greater than 10 s\u22121, all the suspensions had similar viscosities. Conversely, at shear rates less than 10 s\u22121, the viscosity increased with increasing homogenization. There was a similar effect observed with both SIS sheet homogenization and suspension homogenization. Collectively, these findings support that homogenization increased the elastic and thixotropic behavior of the SIS suspensions as indicated by increased crossover strain amplitudes and time-dependent responses. We hypothesize that the protruding fibrillar components of the particles in suspension interact and produce drag between particles that is reflected in the increased elasticity of the suspension. When homogenized suspensions were tested with an ascending strain amplitude, the interaction of the particles overcomes the applied deformation until a sudden breaking point when the particles separate and the suspension flows. When decreasing the strain amplitude, the particles are initially separated by the large strains and are allowed to slowly establish interactions more gradually. Conversely, the particles within non-homogenized samples can slide past each other similarly regardless of the applied strain history because they lack entanglements. These rheological features were then correlated with the electrospun fiber formation of each processing condition to identify key rheological properties that predict spinnability.The bioactivity of dECM scaffolds is strongly dependent on the tissue source of the dECM. Each tissue has a specialized set of proteins, proteoglycans, and growth factors that are tailored to the functions of the tissue [68]. For example, SIS and dermal tissues contain a high concentration of growth factors, such as VEGF, TGF\u03B2, and bFGF, to support a high level of tissue remodeling. Alternatively, cardiac tissue contains an abundance of fibronectin to maintain homeostasis and recruit myofibroblasts for repair [4,68,80,81]. Tissue engineering scaffolds used to treat various tissues and diseases may require a broad range of dECM tissue sources. Thus, the established suspension electrospinning must be functional with many types of dECM. In this study, dECM suspensions were prepared from SIS, skin, and cardiac tissues (Fig. 6). Each suspension was prepared with a dried particle size of 500\u20132000 \u03BCm. For adequate decellularization efficiency, the SIS- and skin-derived ECM were prepared with the Abraham technique, and the heart tissue was prepared with the Badylak technique. To ensure the suspensions had a viscosity that prevented beading, the SIS suspension was prepared with a concentration of 40 mg/mL, the skin suspension was prepared with a concentration of 70 mg/mL, and the Luo suspension was prepared with a concentration of 55 mg/mL. The viscosity flow ramps of the suspensions composed with each dECM tissue source were similar. The ascending crossover strain amplitude of the SIS and skin suspensions were also similar (\u223C400 %); whereas, the heart suspension had an ascending crossover strain amplitude that was significantly lower (\u223C150 %) (Fig. 6C). This indicates that there was reduced particle interaction in the cardiac suspension. Given that these suspensions were all prepared with dual homogenization, these differences were attributed to the specific composition of the cardiac dECM that reduces the particle interaction forces within the suspension. The complex makeup of ECM, including over 600 proteins, makes it difficult to pinpoint specific components that are impacting the overall rheological properties of the ECM composition. Briefly, cardiac-derived ECM has a lower collagen content than intestine- and skin-derived ECM, of which the cardiac-derived ECM contains a higher percentage of non-fibrillar collagen [68]. Fibrillar collagen can form fibril protrusions from the suspended particles and could contribute to the bulk elasticity of the suspension. Additionally, cardiac-derived ECM has been reported to have less fibrillin-2 than intestine- and skin-derived ECM [68]. Fibrillin-2 contributes to the elasticity of tissues allowing them to stretch. In suspension, the presence of fibrillin-2 could contribute to the elevated crossover strain amplitude which facilitates electrospinning. Once electrospun, a mesh composed of continuous fibers was fabricated with each dECM tissue source indicating that a crossover strain amplitude of 150 % is sufficient to maintain elasticity and withstand the electrostatic drawing involved in electrospinning. This further refines the acceptable range of suspension properties for spinnability, where <50 % crossover strain amplitude is primarily beads and >100 % forms continuous fibers when electrospun. Although these suspensions were all spinnable, the fiber morphology differed. The mesh composed of skin-derived ECM contained localized areas of fusion and webbing, and the mesh composed of heart-derived ECM contained flat ribbon-like fibers with a larger fiber diameter than the fibers in the SIS- and skin-based meshes (Fig. S1). Skin tissue has localized areas of hydrophobic and hydrophilic domains to provide a sufficient external barrier and prevent infection [82,83]. The varying compositions may phase separate during homogenization and mixing and cause localized areas of altered fiber morphology. For the cardiac-derived ECM mesh, ribbon formation has been previously attributed to reduced drying of the solvent leading to fiber collapse and the reduced stretching or elasticity of the solution during drawing [49]. Since each suspension contains the same solvent and spinning conditions, it is unlikely that the evaporation rate of HFIP during electrospinning was different between the SIS-, skin-, and heart-derived ECM suspensions. However, the reduced crossover strain amplitude of the heart dECM suspension indicates a reduction of elasticity of the suspension at higher strains. Thus, the suspension was drawn less under electrostatic forces resulting in bigger fibers that dried quickly on the outside and collapsed before the inner fiber could completely dry and retain its shape.The angiogenic capacity of SIS has been widely studied and is one of the primary considerations when selecting SIS as a regenerative scaffold material [5,6,91\u201393]. An in vitro tube formation study and an in ovo chorioallantoic membrane (CAM) assay were both conducted to robustly assess the impact of electrospinning on SIS angiogenic bioactivity. For the tube formation study, HUVECs were cultured on Reduced Growth Factor Matrigel\u00AE in SIS-conditioned media for 4 h (Fig. 8A). The number of capillary-like networks formed in response to the conditioned media was compared between a blank control, a positive VEGF control (20 ng/mL), a decellularized SIS sheet, and an electrospun SIS mesh. All media groups supported some level of tube formation due to the angiogenic cues in Matrigel. However, the increase in networks that formed in response to the positive VEGF control compared to the negative blank control indicated that the network-forming capacity of the HUVECs was not saturated. Further, both SIS substrates supported elevated network formation compared to the blank control and are similar to the VEGF control. Also, the electrospun SIS-conditioned media supported similar network formation to the decellularized SIS sheet-conditioned media. This indicates that the electrospinning process did not negatively impact the angiogenic capacity of the SIS with regard to release products. Since the tube formation assay is limited to the assessment of release products and is artificially inflated by the Matrigel, a CAM assay was conducted for a more robust analysis of the SIS angiogenic capacity. After 10 days of quail embryo culture, a nylon mesh (negative control) [94], decellularized SIS sheet, and electrospun SIS mesh were placed on the CAM for an additional 4 days (Fig. 8B). The change in vasculature was assessed over time with daily imaging. After 4 days of treatment, the primary change in vasculature expected to be seen is small capillary-like vessels, so the vessel density was quantified as the number of vessels intersecting the sample perimeter instead of a vessel area calculation that is heavily skewed by large vessels and sample placement. The Nylon mesh negative control did not significantly impact the vessel density after 4 days on the CAM, indicating that angiogenic cues are needed to increase the vasculature within this timeframe and merely agitation or sample placement on the membrane does not impact angiogenesis. Both of the SIS substrates supported elevated vessel formation compared to the Nylon mesh, and the electrospun SIS mesh supported similar levels of vessel formation compared to the decellularized SIS sheet. Both of the SIS substrates also reduced in size after 4 days on the CAM meaning cells likely migrated into the samples and contracted the ECM. Together, the tube formation assay and CAM assay supported that the angiogenic capacity of the SIS was retained and not negatively impacted after being electrospun.Lastly, the immunomodulatory bioactivity of the electrospun SIS was evaluated via a macrophage polarization assay. The macrophage response to a material has been used to assess whether a material will elicit pro-inflammatory versus regenerative host response [95,96]. For this reason, THP-1-derived macrophages were cultured on the decellularized SIS sheet, electrospun SIS mesh, and a PDMS control. Fold change in gene expression of the macrophages after 72 h of exposure to dECM was quantified in the following genes: NOS2, IL12, CD206, IL10, CHI3L1, VEGF, and TGF\u03B2 (Fig. 8C). The SIS substrates induced a reduction in inflammatory NOS2 and IL12 gene expression and increased gene expression of IL10, CHI3L1, and TGF\u03B2 compared to the M0 naive control. This indicated that SIS substrates drove macrophages toward tolerogenic polarization. The expression of macrophages cultured on the electrospun SIS mesh was largely similar to the decellularized SIS sheet, with statistically similar (p > 0.05) expression of NOS2, IL12, CD206, IL10, CHI3L1, and VEGF. The only gene impacted differentially by the electrospun mesh was TGF\u03B2, where there was a 10-fold increase in TGF\u03B2 expression between the electrospun SIS mesh and the decellularized SIS sheet. Physical characteristics of the electrospun wrap, including the random fiber morphology, are known to alter macrophage activation and could be a potential driving factor in this differential gene expression response [97,98]. TGF\u03B2 plays a role in tissue remodeling by supporting tissue regeneration without scar formation and increasing expression of IL10 and CD206 in macrophages [99,100]. Electrospinning did not largely impact the immunoregulatory bioactivity of SIS via macrophage gene expression with the exception of TGF\u03B2 and supports a tolerogenic and anti-inflammatory immune response at the time point evaluated. Classically pro-inflammatory genes were not induced suggesting that these electrospinning methods do not fundamentally alter the macrophage response to SIS nor is there residual solvent that induce a stress or damage response. Collectively, the angiogenic and immunomodulatory bioactivity of SIS was not significantly impacted by suspension electrospinning. This is largely attributed to the lack of enzymatic digestion and additives required in processing and the retention of protein microstructure above the tertiary organization level.Funding for this work was supported by the National Science Foundation Graduate Research Fellowship Program under Grant (award # 2020300397). Any opinions, findings, conclusions, or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. Funding for this work was supported by the National Science Foundation Graduate Research Fellowship Program under Grant (award # 2020300397). Any opinions, findings, conclusions, or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
PY - 2024/11
Y1 - 2024/11
N2 - Decellularized extracellular matrices (dECM) have strong regenerative potential as tissue engineering scaffolds; however, current clinical options for dECM scaffolds are limited to freeze-drying its native form into sheets. Electrospinning is a versatile scaffold fabrication technique that allows control of macro- and microarchitecture. It remains challenging to electrospin dECM, which has led researchers to either blend it with synthetic materials or use enzymatic digestion to fully solubilize the dECM. Both strategies reduce the innate bioactivity of dECM and limit its regenerative potential. Herein, we developed a new suspension electrospinning method to fabricate a pure dECM fibrous mesh that retains its innate bioactivity. Systematic investigation of suspension parameters was used to identify critical rheological properties required to instill “spinnability,” including homogenization, concentration, and particle size. Homogenization enhanced particle interaction to impart the requisite elastic behavior to withstand electrostatic drawing without breaking. A direct correlation between concentration and viscosity was observed that altered fiber morphology; whereas, particle size had minimal impact on suspension properties and fiber morphology. The versatility of this new method was demonstrated by electrospinning dECM with three common decellularization techniques (Abraham, Badylak, Luo) and tissue sources (intestinal submucosa, heart, skin). Bioactivity retention after electrospinning was confirmed using cell proliferation, angiogenesis, and macrophage polarization assays. Collectively, these findings provide a framework for researchers to electrospin dECM for diverse tissue engineering applications.
AB - Decellularized extracellular matrices (dECM) have strong regenerative potential as tissue engineering scaffolds; however, current clinical options for dECM scaffolds are limited to freeze-drying its native form into sheets. Electrospinning is a versatile scaffold fabrication technique that allows control of macro- and microarchitecture. It remains challenging to electrospin dECM, which has led researchers to either blend it with synthetic materials or use enzymatic digestion to fully solubilize the dECM. Both strategies reduce the innate bioactivity of dECM and limit its regenerative potential. Herein, we developed a new suspension electrospinning method to fabricate a pure dECM fibrous mesh that retains its innate bioactivity. Systematic investigation of suspension parameters was used to identify critical rheological properties required to instill “spinnability,” including homogenization, concentration, and particle size. Homogenization enhanced particle interaction to impart the requisite elastic behavior to withstand electrostatic drawing without breaking. A direct correlation between concentration and viscosity was observed that altered fiber morphology; whereas, particle size had minimal impact on suspension properties and fiber morphology. The versatility of this new method was demonstrated by electrospinning dECM with three common decellularization techniques (Abraham, Badylak, Luo) and tissue sources (intestinal submucosa, heart, skin). Bioactivity retention after electrospinning was confirmed using cell proliferation, angiogenesis, and macrophage polarization assays. Collectively, these findings provide a framework for researchers to electrospin dECM for diverse tissue engineering applications.
KW - Biological scaffolds
KW - Electrospinning
KW - Extracellular matrix
UR - http://www.scopus.com/inward/record.url?scp=85202160352&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85202160352&partnerID=8YFLogxK
U2 - 10.1016/j.bioactmat.2024.08.012
DO - 10.1016/j.bioactmat.2024.08.012
M3 - Article
C2 - 39280898
AN - SCOPUS:85202160352
SN - 2452-199X
VL - 41
SP - 640
EP - 656
JO - Bioactive Materials
JF - Bioactive Materials
ER -