Introduction
Spinal cord injuries (SCIs) are debilitating conditions that affect a patient’s daily activities and quality of life. Estimates have shown that around 20.06 million cases worldwide develop SCIs annually, which has only increased over the last 30 years [1]. The current standard treatment for SCIs focuses on initial resuscitation and stabilization, which is achieved through physical stabilization of the vertebral column and spinal decompression [2]. However, SCIs have a multifaceted pathophysiology that involves inflammation cascade after the initial trauma, which causes damage that lasts until the chronic stage of injury [3]. As such, physicians have adopted a new paradigm called the “Diamond Concept” to account for both mechanical and biological processes occurring during injury by addressing four treatment aspects: structural integrity, neuroconductivity, neuroinductivity, and cellular platform [4].
Cell-based therapies in SCIs are reported to promote axonal regeneration, tissue remodeling, and neovascularization through the production of neurotrophic factors and anti-inflammatory cytokines [3]. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) have been considered one of many options for cellular therapy due to their versatility, availability, and simple harvesting method [5]. However, their application remains limited due to their poor survival rates after transplantation and possible migration outside of the target site. Hence, combination therapy with biomaterials that act as a scaffold could increase their retention and survival rates [6].
Combining natural and synthetic sources is an attractive approach for creating biocompatible scaffolds. Polyvinyl alcohol (PVA) is a synthetic hydrophilic polymer with remarkable biomechanical properties and versatility owing to its modifiable hydrolysis, crystallization rate, and molecular weight [7]. Chitosan (CS), a natural material derived from chitin deacetylation, is known for its biocompatibility and biodegradability, along with additional benefits associated with its antibacterial, antifungal, and analgesic properties [8]. Hence, combining PVA and CS could enhance their scaffold bioproperties, which have been previously found to exhibit versatile functions, particularly in drug delivery and wound dressing [9]. The PVA/CS scaffold has also been found to act as a cellular homing system that promotes regeneration in osteoarthritis [10]. Hence, given its advantageous properties and versatility, the current study aimed to further explore its novel potential in promoting neuroregeneration in SCI by examining the characteristics of the PVA/CS scaffold and evaluating its neuroregeneration potential in canine models.
Materials and Methods
Ethics statement
The study protocol was reviewed and approved by the Ethics Committee of Faculty of Medicine, Public Health, and Nursing (protocol code: KE/FK/1237/EC/2021 and date of approval: November 14, 2021). This study was approved by the Research Ethics Committee of the Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta (approval number: 00134/EC-FKH/Eks./2021). All animal experiments have been conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guideline.
Study design
Mechanical characterization of the PVA/CS scaffold
PVA 10 volume/volume % (v/v%) was diluted in distilled water and mixed using magnetic stirring at 120°C (700 rpm) for 1 hour, whereas CS 11 v/v% was diluted in 2% acetic acid and stirred at 60°C (700 rpm) for 1 hour. The final blend was prepared by mixing both solutions at a PVA:CS ratio of 85:15 referred from a previous study [11]. Afterward, PVA/CS scaffold fibers were prepared using the electrospinning method with an applied voltage of 10–15 kV and a fixed collector distance of 14 cm [12].
Three stages of mechanical testing were conducted sequentially, comparing two different scaffold volumes (6 and 8 mL). Initially, the presence of relevant chemical groups in the produced scaffold was confirmed using Fourier-transform infrared (FTIR) spectroscopy. Afterward, the mechanical strength was assessed using a universal tensile machine (UTM) to determine its maximum deforming force (N). Meanwhile, tensile strength (kPa) was determined by dividing the maximum force by the cross-sectional area, which was expected to resemble the spinal cord mechanical profile of 62.26±5.02 kPa [13]. The surface morphology was visualized under a scanning electron microscope (SEM) and analyzed for scaffold thickness (μm), porosity (%), and nanofiber diameter (nm) using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Biological properties of the PVA/CS scaffold with UC-MSCs
UC-MSCs were obtained from the Stem Cells and Cancer Institute in Jakarta, Indonesia. Isolation and cell culture were conducted at Regenic Laboratory in accordance with Good Manufacturing Practice standards (license number: PW-S.01.04.1.3.333.09.21-0082) and with approval from the Indonesian Food and Drug Administration (Badan Pengawas Obat dan Makanan) and Ministry of Health.
The biodegradation rate was determined as the weight loss percentage after immersing the scaffold in 20 cm3 of phosphate buffer solution (pH 7.4) for 1, 3, 7, 14, and 21 days using the following equation:
where Wm indicates the weights after immersion, Wi denotes the initial weight, and Wloss denotes the percentage of weight loss. Biodegradability half-life was determined when exceeded 50%.
Spectrophotometry was used to evaluate the viability of the cells within the newly established scaffold. Accordingly, PVA/CS scaffolds were placed into 96-well plates (4×4 mm) with a UC-MSC suspension (2.5×104) in each well and were subsequently incubated (37°C, 5% CO2) in 10 μL of MTT reagent for 24 hours. Further, spectrophotometry (550–600 nm) was used to measure absorbance, indicating the number of living cells over several observation periods, i.e., 1, 3, 7, 14, and 21 days. The scaffold was thoroughly washed after every measurement to determine the actual number of proliferations at each observation instance, thereby resembling with the total number of cells on the scaffold surface.
Animal study involving canine SCI models
This study was conducted in accordance with the ARRIVE guideline [14]. Healthy adult canines (Canis Lupus Familiaris) weighing 10–20 kg were included in this study. Animals with musculoskeletal disorders, sensory or autonomic dysfunction, and spinal infection were excluded. Canine models were selected due to their structural and functional similarity to the human spinal cord [15]. The animals were housed in routinely cleaned cages (1×2 m) with padded mattresses.
SCI was induced using the balloon compression method to mimic compression-stimulated SCI [16]. In summary, after the canine models underwent laminotomy, a Fogarty catheter was inserted into the epidural space through the perforated lamina. Next, the balloon was pushed cranially to the level of the T10–T11 segment and then inflated for 6 hours. All the surgical procedures on animals were performed by sedating them with 10 mg/kg body weight (BW) of ketamine intravenously, and was maintained through inhalation of 1%–2% sevoflurane. Later, the balloon was deflated and removed from the epidural space [15].
A total of 12 models were equally divided in random sequence into the following three groups: the control (CD) group receiving no intervention, the mechanical (IM) group receiving only mechanical stabilization (laminectomy, decompression, and stabilization), and the combination (SC) group receiving both mechanical intervention and PVA/CS+UC-MSCs. The sample size was determined based on the resource equation approach for three experimental groups. All interventions were performed by a trained orthopedic surgeon 7 days after balloon compression. Implantation of the scaffold with 106 cells/kg BW was performed by suturing it into the dura mater. All animals were included in the analysis. Evaluations were conducted by two observers and a consensus was reported.
Toxicity evaluation
Animal weights (kg) and laboratory evaluations, which included hemoglobin (Hb), hematocrit (Hct), leukocyte count, aspartate transaminase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine, and urea, were recorded before and after the intervention. The absence of toxicity was confirmed when pre- and post-intervention values did not significantly differ.
Motor improvement
Motor function was assessed using the canine Basso–Beattie–Bresnahan (cBBB) score and recorded over 56 days of observation [17].
Histopathological examination
Euthanasia was performed through propofol injection (6 mg/kg BW) and 20 mg of MgSO4 at the end of the clinical observation. Post-mortem spinal cord specimens were stained with hematoxylin–eosin (HE) stain to investigate hemorrhage and with Luxol fast blue (LFB) stain to visualize myelinization under magnification on the anterior, posterior, and two lateral views of the spinal cord. The scoring system for hemorrhage was as follows: 0=no hemorrhage, 1=scattered ring hemorrhage, 2=coalescing ring hemorrhage and diffuse spread of erythrocytes in the parenchyma, and 3=massive hemorrhage. Meanwhile, scoring for inflammation was as follows: 0=no inflammation, 1=cellular infiltration only in the perivascular areas and meninges, 2=mild cellular infiltration (1/3 of total white matter), and 3=infiltration of inflammatory cells observed in the whole white matter. Scoring for structural damage using the LFB stain was as follows: 1=normally myelinated area, 2=partially demyelinated area (>1/2 cross-sectional intramedullary area), 3=completely demyelinated area, and 4=total structural damage. Three specimen levels were included in the analysis: supralesion (i.e., one level above the lesion), intralesion (i.e., at the level of the lesion), and infralesion (i.e., one level below the lesion).
Statistical analysis
Numeric scales were analyzed using one-way analysis of variance. Meanwhile, ordinal variables were analyzed using the Kruskal-Wallis and Mann-Whitney U post-hoc test. Data normality was assessed using the Sapiro-Wilk test. Statistical analysis was performed using IBM SPSS Statistics ver. 25.0 (2017; IBM Corp., Armonk, NY, USA).
Results
Mechanical characterization of PVA/CS scaffold
Analysis of scaffold structure using FTIR spectroscopy found that relevant functional groups were retained in the scaffold produced by electrospinning, namely the saccharide and amide group of CS (1,152 nm−1 and 1,346 nm−1) and the acetate group of PVA (1,650 nm−1 and 1,150 nm−1). UTM measurements were 159 N and 41.3 kPa for the 6-mL scaffold and 191 N and 49.6 kPa for the 8-mL scaffold. Both volumes demonstrated excellent strength as shown in Fig. 1, where the scaffold remained in the elastic phase before being entirely torn apart, which could be attributed to the mechanic properties of PVA [7]. SEM visualization is depicted in Fig. 2. The 6- and 8-mL scaffold was 171.00±4.72 μm and 228.67±6.01 μm in thickness, 424.67±42.94 nm and 414.33±19.09 nm in fiber diameter, 21.01±1.58 and 21.23±4.33, respectively. Although the 8-mL scaffold showed higher porosity, its production faced syringe blockage, leading to less nanofiber homogeneity. Therefore, 6 mL was determined as the optimal volume for scaffold production.
Biological properties of PVA/CS with UC-MSCs
The half-life of PVA/CS as indicated by >50% scaffold weight loss after immersion with UC-MSCs was reached on day 14 (day 1, 23.01%; day 3, 35.04%; day 7, 42.41%; day 14, 51.81%; day 21, 60.48%). The absorbance value corresponding to the amount of formazan from the MTT assay is presented in Fig. 3. Absorbance values were converted indicating cellular viability peaking at day 14 (day 1, 7,980; day 3, 10,180; day 7, 6,513; day 14, 20,980; day 21, 2,756).
Animal study with canine SCI models
Throughout the observation period, three subjects in the CD group (75%), two subjects in the IM group (50%), and one in the SC group (25%) died. Hence, a total of 12 subjects were included in the statistical analysis. The results of the animal study are presented in Figs. 4 and 5. Survival curves were created using Kaplan-Meier analysis and presented in the Fig. 4A. The evaluation of motor improvement and histopathological examination by the two examiners had an intraclass correlation coefficient of 0.784 (95% confidence interval [CI], 0.76–0.81), indicating that the inter-rater reliability between both observers in this study was good with a p-value of 0.001.
Toxicity evaluation
Weight loss and leukocytosis was observed in all the animals 7 days after the intervention (Table 1). However, no parameter showed a significant difference compared to pre-interventional values, which confirmed the non-toxicity of PVA/CS+UC-MSCs. All laboratory results throuhgout the 7 days after intervention showed that AST and ALP levels, alongside BUN, urea, and creatinine, were within their normal ranges. This finding indicated no metabolic toxicity in the liver and kidneys, especially after the addition of PVA/CS+UC-MSCs in the SC group.
Motor improvement
Differences in motor recovery are illustrated in Fig. 5. Although all groups demonstrated an improvement in the cBBB score, the recovery trend was significantly higher in SC group (pSC-IM=0.012; pSC-CD=0.037). The mean cBBB scores for each group were 0.43, 2.33, and 4.40 for the CD, IM, and SC groups, respectively. The highest cBBB score was different in each group, i.e., 3 (extensive movement of two joints), 7 (extensive movement of all three joints in the HL); and 10 (occasional weight-supported plantar steps; no FL–HL coordination) in CD, IM, and SC group, respectively.
Discussion
Functional recovery of the spinal cord requires comprehensive treatment to address all four pillars of the Diamond Concept. Stem cell therapy could provide a cellular platform with neuroinductive cytokines in combination with the routine surgical treatment to address structural integrity. However, cell-based therapies require a proper microenvironment for the cells to thrive. Combination therapy with biomaterial scaffolds could facilitate such a treatment approach by acting as the base for cell infiltration and directing the treatment effects to the target area [4] (Fig. 6).
Various materials have been proposed for the creation of these stem cell-supporting scaffolds. Composites of several polymers have been the typical approach [18]. Ideally, scaffolds should consist of robust media with adequate biocompatibility to support cellular bioactivity [3], which is achieved by combining the robust PVA polymers with natural biological properties, such as that in CS [19]. Scaffold characterization was conducted to ensure that the scaffold had nano-sized diameters, which function to provide a large surface-to-volume ratio, and appropriate porosity to allow biomolecule diffusion while still limiting the inflammatory cell infiltration. These properties enable a stable microenvironment for cellular viability [20,21]. Additionally, PVA/CS is also unique in its increased tensile strength and structural stability in liquid environments, making it suitable for intracanal implantation [18,22].
Our study showed certain biomolecular reactions toward PVA/CS contact. Notably, we found decreased proliferation rates in at day 7, which then increased at day 14. This finding suggests that the presence of biomaterial contact can affect the proliferation process. Similarly, one previous study using UC-MSCs and synthetic scaffolds showed a decrease in proliferation at certain days of observation [23]. Another study attributes this phenomenon to contact inhibition factors expressed by stem cells on contact with new biomaterials. As shown in Fig. 4, the SC group had the highest cBBB score improvement on the day 14, suggesting neuroregeneration as indicated by the return of motor function.
Neuronal regeneration was demonstrated by the improvement in motor function and histopathological examination after 56 days of observation, which encompasses the acute and subacute phases of SCI pathophysiology, during which the most significant biological recovery occurs [24,25]. Previous studies have reported similar results using various scaffold materials in comparison with cellular therapy alone [26,27]. However, the current study also highlighted the superiority of combination therapy when compared to mechanical intervention group, which represents the routine therapeutic recommendation for SCI.
Notably, MSCs promote neuronal recovery through their immunomodulatory effects, which reduce the pro-inflammatory microglia (M1) frequency, activate their anti-inflammatory phenotype (M2) through the secretion of interleukin (IL)-4 and IL-13, and reduce tumor necrosis factor-α and IL-6 [28,29]. Generally, inflammatory responses following SCI have played a role in further aggravating the damage after the initial trauma [30]. In present study, this mechanism has been shown to promote a healing environment that facilitates axonal growth and functional recovery as shown by the decreased demyelination on LFB staining. Meanwhile, scaffolding promotes neuronal recovery by decreasing vacuole formation and tissue cavitation, thereby increasing neuronal structural density in the injured spinal cord. This finding suggests that a combination of scaffold and stem cell therapy inhibits glial reaction and prevents the formation of glial scars Although glial scars act as a natural protective barrier in the acute phase of SCI, it significantly inhibits neuronal growth, with its absence allowing for tissue regeneration [4].
A high mortality rate was found in the groups that did not receive stem cell therapy, which might indicate the potential positive effects of stem cells on survival rate in SCI, although further studies are necessary to confirm this finding. Mortality was also suspected to be caused by sepsis from pressure ulcers associated with the limited mobilization of the subjects, which was a limitation of this study. The affected subjects were still included for statistical analysis.
Overall, our findings support the incorporation of stem cell–scaffold therapy to routine mechanical treatment, which can provide hope for functional return for SCI patients. However, longer observations of up to 90–120 days are warranted to determine the effects of this combination therapy in the chronic phase of injury. Nonhuman primate models could also be used to further validate the effects of stem cells, although ethical issues should be taken into consideration.
Conclusions
Neuroregeneration in SCI could be achieved by reinforcing all four pillars of the Diamond Concept. PVA/CS scaffolds could act as a homing system for cellular therapy in SCI with excellent mechanical and biological properties, which had been confirmed by the clinical and histopathological improvements in our canine SCI models after combining it with UCS-MSCs in addition to mechanical intervention.
Key Points
The mechanical profile of the polyvinyl alcohol/chitosan (PVA/CS) scaffold showed relevant chemical groups, nano-sized diameters with an average of 434.88±24.39 nm, and excellent strength profile (159 N; 41.3 kPa).
Biological tests showed stem cell biocompatibility to the PVA/CS scaffold with a biodegradation half-life of 14 days.
Significant motor improvements were observed in canine spinal cord injury (SCI) models receiving PVA/CS–umbilical cord-derived mesenchymal stem cells intervention (SC) compared to those receiving the control and mechanical intervention (pCD=0.037; pIM=0.012).
Our clinical findings were supported by histopathological findings, which showing that the SC group exhibited significantly less intralesional hemorrhage (pCD=0.020; pIM=0.016) and less demyelination with Luxol fast blue staining on the anterior (pCD=0.020; pIM=0.016), lateral 1 (pCD=0.012; pIM=0.048), and lateral 2 (pCD=0.007; pIM=0.027) views of the spinal cord.
PVA/CS scaffolds, which act as a homing system for stem cells, promote neuroregeneration, and could serve as further comprehensive treatment of SCI in conjunction with routine stabilization therapy.
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