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Introduction

Short bowel syndrome (SBS) is a rare disorder characterised by malabsorption of nutrients because of severe bowel loss. After losing an extensive small bowel length, the patient develops gastrointestinal (GI) symptoms such as diarrhoea, steatorrhea, weight loss, malnutrition, and dehydration. Short bowel syndrome is the leading cause of Intestinal Failure (IF), and it involves the need for long- term parenteral nutrition (PN) to ensure patients nutritional needs-4. Short bowel syndrome is a multi-systemic disease, and its treatment entails multidisciplinary teams involving surgeons, dieticians, gastroenterologists, specialised nurses, and psychologists. Nevertheless, overall survival has increased over the last decades, mainly due to improvements in medical and surgical treatment strategies and new PN formulas^.

The most frequent occurrences that bring to the subtotal loss of the small intestine in early life are necrotising enterocolitis (NEC), small intestinal volvulus, gastroschisis and apple peel intestinal atresia. Other rare conditions, such as incarcerated congenital diaphragmatic hernia, have also been reported ink. Early surgical intervention should focus on preserving bowel length in all these cases. Short bowel syndrome is a devastating disorder that profoundly affects childrens and their families lives. Complications of IF are severe and still common among SBS patients despite the establishment of innovative Intestinal Rehabilitation Programs. Sepsis from central line infections and intestinal failure-associated liver disease (IFALD) are the most frequent conditions influencing patient outcomes. Furthermore, autologous gastro- intestinal reconstruction (AGIR) techniques have high mortality and morbidity, and managing their complications is difficult and expensive 7 .

New techniques have been studied to improve intestinal length in SBS and IF patients  since the  end of the past century-It is well known that biological systems and tissues such as skin, bone and lungs react to mechanical strengths. Therefore, mechanical distraction is routinely used in different clinical contexts,e.g., bone lengthening by distraction osteogenesis or skin growth by tissue expansion. Distraction enterogenesis (DE) is based on applying linear longitudinal traction in different isolated bowel loops to stimulate the tissue’s growth and proliferation and promote intestinal lengthening 3 ^ Several devices and materials have been proposed as a means of intestinal distraction; Okawada and colleagues 5 created a murine model of DE usinp an infusion of hiph molecular weipht polyethene plycol (PEG) in isolated intestinal loops. Polyethene plycol is an osmotic laxative that induces bowel dilation by drawing liquids in the intestinal lumen 7  5. In addition, Fisher et a/. designed a shape-memory polymer cylinder positioned extraluminally that self-expands causing bowel distraction*.

Later, further studies introduced wholly implantable intraluminal devices made of different materials such as nitinol and polycaprolactone (PCL). The implantation of these biocompatible sprints inside the lumen assumes a surgical intervention or an endoscopic procedure. In addition, if the material used is not biodegradable, a second intervention will be necessary to remove it after the distraction process is considered satisfactory. Distraction enteropenesis has also been shown to induce adaptive histolopic changes e.p., thickening of the muscular layer and deepening of the crypts; mechanisms at the base of those events still need to be well known.This study discusses the possibility of usinp biodegradable materials (e.p., silk fibroin and gelatine) to convey devices that facilitate intestinal prowth. These devices could be used in new mini-invasive DE techniques. As proof of this concept, we consider usinp gelatine capsules modified with silk fibroin as carriers for 3D-printed sprints.

Materials and methods

The study aimed to create edible capsules that can be used as a vehicle for drugs and or devices that can enhance intestinal prowth in specific intestine tracts. To reach their tarpet, these capsules should be biodegradable, soluble in water, resistant to degradation in acidic conditions and simple to create.
The choice of materials used to produce the capsules was based on their biocompatibility, biodepradability, and solubility 2 2. After creation, the capsules were tested to check their resistance to degradation in acidic solutions usinp Phosphate Buffered Saline (PBS), composed of Calcium chloride and Magnesium chloride (CaCl2+ MgCl2) with a pH value of 7.4 and acetic acid (CH3COOH) with a pH value of 2.3, both at 35 to recreate the conditions found in the gastrointestinal tract. The capsule resistance to degradation was studied using the Fourier-transform infrared spectroscopy (FTIR) and analysing their weight before and after immersion.

The analysis of resistance to degradation required 1-minute immersions, a drying process and weight assessment for gelatine-only and gelatine and fibroin capsules. Weight variation was calculated using the formula: [(Final weight - Initial weight)/Initial weight] *100. In this study, the capsules were used as vehicles for 3D-printed sprints, which are used for the distraction enteropenesis process in SBS patients. The sprints expansion process is a function of the degradation of the capsules in which the sprints are contained. Therefore, our tests were focused on the timinp of sprinp release both in gastric and intestinal environments; obtained results were then compared with the elastic constant of the encapsulated sprinp. Capsules were first realised with a solution of 10% gelatin (pigskin type A from Merck) mixed in 20 ml of distilled water. To create a homogeneous solution, magnetic stirrers were used at 60; after 45 minutes, we obtained a complete dissolution of the gelatin, which was then poured into the mould (Figure 1a). Moulds were designed with 3 pairs of elliptical holes, 0.6 cm deep, with dimensions of 1.1 cm x 0.5 cm, 1.1 cm x 0.4 cm and 0.9 cm x 0.4 cm, respectively.

Capsules are obtained by pouring the solutions into the moulds (Figure 1B) and will subsequently be analysed and characterised. To obtain more excellent resistance to degradation in acidic solutions, regenerated silk fibroin was added to the solution - the presence of gelatin grants further protection from light and oxygen23. The gelatin and regenerated silk fibroin mixture can change the hydrophilic interactions between silk and water, thus creating a more stable and homogeneous system by creating porous structures. Bombyx mon silk cocoons (10g) were boiled for 30 minutes in 200ml of water containing 5g NaHCO3. The extract fibres were washed two times with water and dried at room temperature under a chemical hood in laminar flow. Subsequently, the fibroin fibres were dissolved in Sml of FA containing an amount of CaCI2 equal to 60:40 concerning the weipht of the dried fibroin fibres (i.e. 0.65p) at 30C for 1 hour (e.g. 0.43p).

The compound thus obtained is dissolved distilled water to get a 20 ml solution to which 10% gelatin is added. The solution is placed on a 5 cm Petri dish to let the solvent overnight and then put at 40 for 4 hours. Finally, the solution is homopenised usinp magnetic agitators at 60 for 30 minutes and then poured into a mould and left to solidify for 6 days (Figure 1).

Fig. 1. (A) Picture of the mould used to realise the capsules and (B) picture of the capsules of different shapes and dimensions acer the extraction from the mould. The scale bar indicates I cm.

Springs are composed of a structure of regenerated silk (RS) modified with praphene nanoplatelets (GNP) externally covered with a biodegradable polyhydroxybutyrate-valerate (PHBV) shell2^. Briefly, sprints were realised with 3D printing, through which, with an extruder, PHBV and RS compounds are deposited simultaneously in a 3D structure with the addition of praphene nanoplatelets (GN Ps) to the RS25. Polyhydroxybutyrate- valerate was chosen because it is a non- cytotoxic biocompatible polymer suitable for in vivo use. Table 1 shows the details of the solution of RS used for 3D printing.

Table l. RS solutions are used for 3D printing.

Springs were inserted into the moulds before phase, the length of which was defined pouring the RS and gelatine solution to through trial and error  encapsulate them with the solidification (Figures 2 A and 2 B).

Fig. 2. (A) Springs inserted in the mould and (B) lateral view of the springs inside the solution of gelatine and silk fibroin. The scale bar indicates cm.

If the process lasts lonper than 3 days, the solution alters, makinp it useless. Figure 3 shows a sprinp inside a capsule after the solidification phase.

Results

As mentioned above, the stability of gelatine- only and gelatine and fibroin capsules was five minutes. Gelatine-only capsules showed a rapid degradation time with total weight loss after tested by submerging them in 10 ml of PBS.

Figure 4. Degradation progress sequence of gelatine-only capsules in PBS 2.

On the other hand, gelatine and fibroin moulds containing type 3 and 4 springs were capsules proved to be more resistant, with a tested in both fluids instead. degradation time of 45 minutes (Figure 4). We also analysed this compound in acetic acid (pH 2.3), simulating the gastric environment with a resistance of over 90 minutes. These results should be considered satisfying considering the medium gastric and intestinal transit time2^.

In both experiments, we observed an initial prowth in the weipht and size of the capsules; a starting PBS absorption can explain this by the capsules themselves. Once we established that the gelatine and fibroin capsules were the most stable choice, we analysed the releasing times of the sprints in pH-controlled environments. The dissolution tests were conducted at 35’C, submerpinp the capsules in 10 ml of PBS or acetic acid to simulate intestinal and gastric environments. Capsules obtained in 1.1 x 0.5 cm moulds containing type 1 e 2 springs were studied in acetic acid and PBS, respectively; capsules obtained in 1.1 x 0.4 cm and 0.9 x 0.4 cm

For example, we report in Figure 7 the degradation process of the capsule containing type 1 springs with a release time of around 1 20 min.

Figure 5. Photographic sequence of dissolution test of the capsule containing type! spring in acetic acid.

Results shown in Table 2 and Figure 6 suggest that the pH plays a vital role in the correlation between the elastic constant of the springs and the releasing time. The releasing time grows with the increase of the elastic constant when the capsules are tested in acetic acid (e.g., simulated gastric fluid (SGF)). At the same time, the opposite happens in PBS (e.g., simulated intestine fluid (SIF)). These results must be confirmed with in vivo experiments.

Figure b. Values of the elastic constant of the springs according to the dissolution time in RS gelatine capsules.

Table 2. Degradation times of RS gelatine capsules as regards the elastic constant of the 3D coated springs.

Data from Figure 8 and Table 2 show that while carrying the springs, gelatine and fibroin capsules releasing times are compatible with gastric and intestinal transit time in PBS and acetic acid.

Discussion

Surgical treatment of Short bowel syndrome focuses on lengthening and remodelling procedures to restore intestinal function, peristalsis, calibre, and length. Non-transplant surgery in SBS focuses on the principle of Autologous Gastrointestinal Reconstruction procedures (AGIR): controlled bowel expansion 27 28, lengthening and tailoring procedures such as Longitudinal Intestinal Lenghtening and Tailoring (LILT), Serial Transverse  Enteroplasty (STEP)  and Spiral Intestinal  Lenghtening  and Tailoring (SILT)2- 31d slowing transit time using reverse segments or colonic interposition 3. Sometimes more than one surgical procedure is required to achieve enteral autonomy, together with medical and nutritional management 33. Unfortunately, often these techniques are not enough to obtain weaning from PN. New frontiers are continuously researched in medical management, such as GLP-2 treatments, new PN formulas and surgical side, such as organoids 34 35 and distraction enterogenesis. Distraction enterogenesis is a promising method for less invasive treatment of SBS of increasing intestinal length.

A few studies have been conducted so far to demonstrate the effectiveness of this method on pre-clinical and in vivo animal models3 3. Our study focused on the possibility of creating edible capsules that could reach the human intestine and release 3D-printed sprints. The first step required a careful choice of materials. Initially, capsules were realised only usinp gelatine from pipskin type A from Sipma-Aldrich, which is already broadly used in the pharmaceutical industry to produce edible capsules. The low resistance to degradation in PBS and subsequently in the intestinal tract at a pH of 7.4 brought us to combine gelatine and RS to realise our capsules. We conducted different experiments with various compositions and concentration levels to define the perfect mixture. This new compound showed an optimal resistance to degradation both in gastric and intestinal environments, with a substantial increase in timings of dissolution compared to gelatine alone. Analysis in acidic and basic conditions confirmed the stability of the solution with a transit time that could allow the capsule to reach the intestine without dissolving (Table 1). After testing the capsules’ resistance, we combined them with 3D-printed sprints, confirming the potential ability of the device to reach the intestine and release the 3D sprints. Durinp this phase, we noted variability in release timings as a function of the pH value to which the capsules were exposed, and the elastic constants of the sprinp contained. When immersed in acetic acid, the release time prew following the elastic constant of the sprinp, while in PBS, the opposite trend was observed.

Our work is a preliminary study for the realisation of biocompatible devices that could carry endoluminal implantable 3D structures and drugs or growth factors in the human intestine, setting the foundation for a new approach to distraction enterogenesis for the treatment of SBS patients. With this new approach, we could deliver scaffolds enriched with growth factors into the intestine to enhance cellular proliferation, treat ulcers or anastomosis leaks, or even delay surgical treatment in selected patients. The capsules must be tested in vivo in animals to study the sprints feasibility and correct positioning to reach these poals. Springs need specific characteristics of diameter, lenpth, and elastic constants that need to be correlated with intestinal dimensions 8 3 . The poal is to have enough elastic strength to determine intestinal lenptheninp without causing damape. From previous studies, it has been shown that the relationship between the elastic strength and the weipht of the animal should be better at 0.83 and 0.117 3 4*.

In our study, the elastic constants ranpe from 1.10 ± 0.01 N/m to 1.60 ± 0.01 N/m, similar to what was achieved in rats by Sullins et al 1 With these capsules, the sprints would reach the intestine without surgery. However, subsequently, they will not have a secure attachment to the intestinal wall, which could lower their ability to exert the traction necessary for distraction enteropenesis. Previous studies have tried usinp hiph-friction materials to overcome this problem and diminish dislodpement risk. Recent studies solved this problem by altering the 3D structure of the sprinp and addinp anchoring devices to the external surface, which allows a safer attachment of the sprints to the intestinal wall. In further experiments, our 3D-printed sprints could be modified in the desipn phase to add these anchoring structures for more stability.

Conclusions

This preliminary study showed that capsules realised from gelatine and RS are resistant to degradation in the intestinal environment, possibly carrying 3D springs, drugs or other devices in specific intestine tracts without the need for surgery. These devices could greatly assist the distraction enterogenesis processes for treating SBS patients. However, further animal in also studies are needed to better understand the springs feasibility and correct positioning.
 
Corresponding Author: Francesca Gigola
Meyer Childrens Hospital IRCCS Viale Pieraccini 24, Florence, Italy Email: [email protected]

Conflicts of Interests: None.

Funding Statement: None

Acknowledgement None

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