Poly(lactic-acid) (PLA) and poly(ε-caprolactone) (PCL) are also termed as ‘spine polymers’, due to their abundant use in spinal implants and related applications. Approved for hard- and soft-tissue repair by the U.S. Food and Drug Administration, PLA and PCL have been employed in several orthopedic implants and disc disorder treatments20,21,22,23,24. The slow-degrading PCL and fast-degrading PLA can be blended to attain excellent mechanical properties, shape memory characteristics and biocompatibility with reduced inflammatory response25,26,27,28. Our designed patch comprises of a PLA-PCL blend14, and the in-vitro degradation behavior will be explored in this study.
Biodegradable polymers employed in the designed patch enable gradual degradation after implantation, preventing costs and trauma associated with secondary patch removal procedures. The degradation scheme involves aqueous diffusion in the polymer matrix followed by hydrolytic degradation by water or enzymes. Ester group hydrolysis leads to chain scission and the formation of carboxylic and alcoholic end groups18,22. The designed patch is composed of a PLA-PCL blend, with the two polymers mixed in 83:17 weight ratios respectively14. A minor amount of PCL is known to accelerate the PLA hydrolysis in blend films, due to an increased concentration of terminal carboxyl groups29. To study in-vitro degradation, the designed patches were exposed to phosphate buffered saline (average pH 7.4) and amniotic fluid (average pH 8.26) separately. Amniotic fluid contains enzymes with varying activity levels based on the gestation period, however, there is no evidence of their participation in ester hydrolysis30,31. Our study did not attempt to investigate enzyme activity in the amniotic fluid used32,33.
Previous work conducted by our group documents the effect of in-vitro degradation on weight loss, surface roughness and functional groups14. It is critical to discuss these properties in this section as they corroborate the findings from crystallinity and mechanical property investigations and provide a holistic understanding of the in-vitro degradation profile. The trend of weight loss experienced by the designed patch was observed in earlier studies of PLA-PCL blends14,29. Basic media is known to accelerate the degradation, caused by more increased availability of base attack sites18. However, the difference in pH of PBS and AF did not significantly affect the weight loss at 16 weeks14. Thus, the presence of enzymes in AF failed to impact bulk degradation to the extent of impacting weight loss.
Modification of surface properties can influence the hydrolytic degradation of aliphatic polyesters29. Enzymes present in media modify the surface and physico-chemical characteristics31 but fail to impact the breakdown of PCL on implantation34. Surface erosion is a strong indicator of hydrolysis in PLA-based systems, corroborated by literature reporting a linear relationship between surface roughness and time on aqueous exposure35,36. From previous work conducted on the designed patches, amniotic fluid exposure caused greater changes in surface roughness compared to phosphate-buffered saline at 16 weeks14. Atomic force microscopy images taken at 16 weeks of AF exposure exhibited larger diameter surface craters, indicating the extent of surface impact and hydrolysis of susceptible PLA segments.
The chemical interactions in PLA-PCL blends are caused by hydrogen bonding between C=O group in PCL and terminal hydroxyl groups in PLA37. Polyester films immersed in basic aqueous solutions display absorbance peaks at 1570 cm−1, as reported in literature38. Similarly, exposure to phosphate-buffered saline causes an increase in area of peak at 1750 cm−1. This indicates greater number of carbonyl bonds, which are part of the terminal [COOH] end-group. A greater carboxylic acid end-group concentration results from chain scission during hydrolysis of PLA and PCL39. Accelerated hydrolysis takes place due to rising hydrophilicity, as more esters are converted to acid and alcohol end-groups40.
The participation of amniotic fluid enzymes in bulk ester hydrolysis has not been reported, however, the enzymes might impact the surface properties31. From previous work conducted on the designed patch, amniotic fluid exposure exhibited presence of carbonyl peaks and visible stretching of O–H peak at 16 weeks, compared to 16 weeks spectra on PBS exposure where minimal changes in the O–H peak were observed14. These findings indicate more terminal hydroxyl groups in the system, which can advance the rate of hydrolysis. Acceleration of relative hydrolytic surface degradation has been evidenced on exposure to basic media in other lower molecular weight lactide-based systems and might be similarly impacting the PLA domains in the designed patch39. Similar to the percolation phenomenon, the aqueous media reaches the bulk of the system only at 16 weeks, triggering the release of degradation products and creation of hydroxyl groups40.
Both PLA and PCL consist of crystalline and amorphous domains in varying content, based on molecular weight and crystallinity (Supplementary Fig. 2). However, it should be noted that PLA domains are major contributors to the crystalline content of the blend, owing to the low molecular weight and percentage of the PCL used in the blend. Studying the changes in crystallinity provides valuable information about internal rearrangements in the blend structure and can offer deep insight into the degradation behavior. Hydrolytic degradation is expedited by oligomeric products such as carboxylic acids within the blend matrix41. The increasing content of carboxylic acid-containing oligomers has been confirmed by results of FTIR-ATR tests from the previous study14.
On analyzing XRD data of PBS exposure, the disappearance of the amorphous region between 14° and 16° beyond the 4-week time point indicates bulk erosion and redistribution of domains. This occurrence at the 4-week time point can be linked to the initial penetration of aqueous media in the bulk of the system, followed by displacement of oligomeric products outwards from the core. Between the 4-week and 8-week time points, the exposure to 37 °C establishes chain mobility in PCL, triggering the recrystallization of these oligomeric products. This translates into relatively crystalline peaks observed at 8, 12 and 16-week time points.
XRD results on amniotic fluid exposure display a pronounced increase in crystalline domains at the 16-week time point. This can be noticed by the narrower peaks noticed at 16-week time point in comparison to peaks at the remaining time points, seen at 17.41°, 19.60°, 21.51° and 23.92° 2theta. The half-width of peaks relates to the crystallite dimensions, and the narrow half-width of peaks observed at 16-week exposure to amniotic fluid indicates the existence of large crystallites. The exposure to aqueous media at 37 °C leads to an annealing effect that contributes to the ordered arrangement of chains42. The increase in crystallinity of PCL domains can also be attributed to degradation-induced crystallization of amorphous domains in the matrix43. Pitt et al. reported a consistent but slower crystallinity increase beyond 4 weeks of PCL implantation in a rabbit model, which can be linked to crystallization of tie segments facilitated by chain cleavage in amorphous phase because of low glass transition temperature (− 60 °C) of PCL42. Similarly, the combined effect of temperature on PCL, and enzymes on PLA, accelerates the surface erosion of respective low molecular weight fractions, also supported by findings from surface roughness studies14. At the 16-week time point on XRD data, amorphous fractions are also visible between 12° and 16°, and an amorphous component is observed between 22° and 23°. This finding indicates the sequential separation of amorphous domains taking place alongside the formation of large crystallites in the blend matrix. This argument is supported by the ATR-FTIR data at 16 weeks of amniotic fluid exposure, which reports more terminal hydroxyl groups due to release of degradation products in the system14.
The change in mechanical integrity is a strong indicator of degradation in polymer blends. Brittle behavior at 16 weeks for PLA-PCL blends has been reported in previously published studies41. This phenomenon is also visible in the x-ray diffraction studies, where the crystalline peaks are retained at 16 weeks, even though the specimens have undergone considerable strength loss compared to the control (0 weeks) specimens. Additionally, the relative narrowing of peaks observed at the 16-week time point of amniotic fluid exposure consolidates the transition to brittle behavior.
Polymer chains react at a slow rate during hydrolysis, leading to chain cleavage and added mobility. This causes a gradual and prominent increase in crystalline domains, that eventually impacts mechanical integrity. Poly(lactic acid) exposure to a temperature of 37 °C creates an annealing effect that also impacts crystallinity. Due to this effect, the amorphous domains produce a spherulite microstructure consisting ordered lamellae inter-connected by short chains. Depending on the time of annealing, the spherulites occupy partial or complete volume of the polymer. This leads to two configurations of the amorphous domains: (a) fully grown spherulites and (b) partially grown spherulites with remaining volume occupied by ordered lamellae44. Tsuji et al. proposed that ordered lamellae inter-connected by short chains might have a higher density of terminal carboxyl and hydroxyl groups that do not participate in crystallization. This suggests that the two configurations of amorphous domains in PLA matrix can degrade at different rates, which might impact the crystallinity and mechanical properties45.
The low glass transition temperature of PCL (− 60 °C) could lead to significant chain mobility due to annealing. The chain mobility would rise further due to reduction of chain length during hydrolysis. This can lead to recrystallization and substantially contribute to the crystallinity of the polymer blend46. The impact of annealing on crystallinity of the blend can be directly linked to the mechanical behavior. When the blend displays ductile behavior, it indicates the presence of disoriented amorphous domains that can be further oriented into crystalline structures on the application of strain. However, the transition of ductile to brittle behavior signifies negligible disorientation in the blend, and an optimal content of crystalline domains.
As the designed patch is unique in terms of the composition and functional properties, it is less likely to conduct an adequate comparison to commercialized products individually, as the new patch collectively possesses the claimed features that are superior to one or certain, if not all, unmet needs of the conventional patches. For example, cellulose-based patches are known to be porous, indicating the permeability will be different compared to the designed patch and thus comparative mechanical testing will not lead to a uniform comparison. However, mechanical properties of the designed patch were compared to values reported in literature to understand the range of mechanical response. The designed patch has modulus values that are much higher than commercially used patches, while the tensile strength is higher, and in some cases lower than the commercially used patches47. However, the key metric for the suitability of an artificial fetal MMC patch is the similarity to mechanical properties of cranial human dura matter. The tensile strength of the designed patches was similar to that of cranial human dura matter47 and remained stable during 16 weeks of exposure in PBS and amniotic fluid.
Due to unavailability of published data on polymer degradation in amniotic fluid, phosphate-buffered saline was used as a reference, and also to emulate body fluids. Degradation of different polymer systems in PBS has been studied before, but the degradation kinetics of our PLA-PCL blend needed to be investigated when subjected to simulated fetal conditions of movements and temperature. The continuous rotations and vibrations will cause rigorous movement of fluid in the tubes, which would strongly interact with the immersed patch strips. Due to these interactions, the fluid might generate a cyclic stress on the patch, that can influence the rate of hydrolysis.
On review of mechanical testing results, exposure to both media, i.e. phosphate-buffered saline and amniotic fluid, led to a brittle fracture of the PLA-PCL blend at 16 weeks. Though the mechanical integrity of the patch does not deteriorate at 16 weeks, all the above characterizations indicate the progress of hydrolytic degradation. Lastly, crack initiation and physical disintegration of additional sets of patches observed at 20 weeks of PBS and AF exposure consolidate the in-vitro biodegradability of the designed patch. This can be an effect of the simulated fetal environment and the interaction with the amniotic fluid cells, causing accelerated disintegration of the patch. Our previous experiments in the PLA/PCL patch showed biocompatibility as a cell substrate in vitro that could help during the healing processes in vivo14 but also affect the degradation of the designated patch. Further studies are needed to evaluate the degradation effect and cell interaction.
The onset of brittle behavior from 12 weeks of amniotic fluid exposure and 16 weeks of phosphate-buffered saline is evident from the mechanical testing data. This does not comply well with the flexibility required in the patch to incorporate the radial expansion during fetal growth. However, an accurate mechanical response of the designed patch can be gauged via in-vivo implantation in animal models, where the patch would simultaneously experience contact with physiological fluid and amniotic fluid, in addition to temperature and fetal movements. Modifications in the blending technique and addition of branching agents to improve the flexibility of the patch will be explored, which will help enhance the comfort of the surgeon and compliance of the patch to adapt to the spinal canal during prenatal repair of spina bifida.
