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A long-lasting guided bone regeneration membrane from sequentially functionalised photoactive atelocollagen

The fast degradation of collagen-based membranes in the biological environment remains a critical challenge, resulting in underperforming Guided Bone Regeneration (GBR) therapy leading to compromised clinical results. Photoactive atelocollagen (AC) systems functionalised with ethylenically unsaturated monomers, such as 4-vinylbenzyl chloride (4VBC), have been shown to generate mechanically competent materials for wound healing, inflammation control and drug delivery, whereby control of the molecular architecture of the AC network is key. Building on this platform, the sequential functionalisation with 4VBC and methacrylic anhydride (MA) was hypothesised to generate UV-cured AC hydrogels with reduced swelling ratio, increased proteolytic stability and barrier functionality for GBR therapy. The sequentially functionalised atelocollagen precursor (SAP) was characterised via TNBS and ninhydrin colourimetric assays, circular dichroism and UV-curing rheometry, which confirmed nearly complete consumption of collagen primary amino groups, preserved triple helices and fast (within 180 s) gelation kinetics, respectively. Hydrogel swelling ratio and compression modulus were adjusted depending on the aqueous environment used for UV-curing, whilst the sequential functionalisation of AC successfully generated hydrogels with superior proteolytic stability in vitro compared to both 4VBC functionalised control and the commercial dental membrane Bio-Gide. These in vitro results were confirmed in vivo via both subcutaneous implantation and a proof-of-concept study in a GBR calvarial model, indicating integrity of the hydrogel and barrier defect, as well as tissue formation following 1-month implantation in rats.


Introduction
Guided Bone Regeneration (GBR) therapy has gained increasing attention, due to the growing trends in bone disorders associated with ageing, congenital diseases, trauma, and cancer, and the avoidance of tissue morbidity commonly observed with autologous bone grafting [1,2]. GBR therapy has proven successful in supporting the repair of alveolar bone critical-sized defects in the oral cavity [3], whilst new GBR strategies integrating membranes and soluble factors have also been explored for orthopaedic reconstruction [ 4 , 5 ]. An essential component of GBR therapy in dentistry is the placement of a physical barrier between the connective tissue and the critical-sized alveolar bone defect to allow repopulation of region-specific osteogenic cells in the bone defect [6,7]. The GBR membrane acts as a barrier against soft tissue cells present in the connective tissue, aiming to promote the selective proliferation of osteogenic cells. Together with the GBR membrane, a graft material may also be applied to the critical-sized bone defect in vivo to provide an osteoconductive environment for bone regeneration. In addition, the graft material may also be applied to ensure membrane conformation to, and preservation of, the critical-sized defect, minimising risks of membrane collapse into the defect [8,9].
Resorption of the GBR membrane in vivo is a key aim in promoting full bone regeneration with a single-step clinical procedure, minimising the infection risks associated with a second surgery and membrane removal [10]. Although an ideal degradation profile has yet to be established, it is well accepted that the membrane must retain its barrier function for a certain amount of time following implantation in vivo to achieve a predictable regenerative outcome [11,12]. Considering that completed bone regeneration can take up to six months depending on the defect size [13], it can be estimated that the GBR membrane should display material integrity in the range from four weeks, when the bone remodelling phase starts, up to several months, to preserve the volume of the bone tissue defect, support selective homing of osteogenic cells and avoid soft tissue infiltration. Overall, the membrane should display (i) elasticity, to enable clinical handling and implantation in vivo with no mechanical damage; (ii) space-maintaining capability, to ensure conformation with, and protection of, the bone defect; (iii) tissue occlusivity, to minimise risks of soft tissue infiltration following implantation and during degradation; (iv) safe degradability to minimise risks of eliciting toxic response during membrane degradation [14]. Although GBR membranes have been used widely to treat bone defects in relation to severe periodontitis, peri-implantitis, and large bone loss, long term stability in vivo (> 1 month) is still a challenge for most clinically approved resorbable GBR products [14,15,16,17], resulting in soft tissue ingrowth and underperforming clinical outcome [18].
Multiple natural polymers, e.g. bacterial cellulose [2], chitin [19], hyaluronic acid [20], poly(-glutamic acid) [21], and silk [22], have recently been proposed as protease-insensitive substrates for the design of new resorbable GBR membranes with prolonged stability in the physiological environment. Customisation of multiphase formulations into bespoke fibrous configurations has proven promising, aiming to induce membrane tissue adhesion, trigger an osteogenic environment in vitro and minimise risks of surgery-induced infection [6,22,23,24,25]. These approaches have subsequently been translated to synthetic polymers aiming to overcome the potential toxicity of degradation products [17], and to accomplish mechanical competence and space maintenance capability, together with osteogenic and barrier functionalities in vitro [26,27,28]. While these research efforts have shown promise at the preclinical testing stage, clinically approved resorbable membranes are still mainly made of type I collagen [7,14,18]. This is largely attributed to the fact that type I collagen is the main protein component of the extracellular matrix (ECM) of bone, whereby the hierarchical structure and natural crosslinks of collagen regulate bone function and remodelling in vivo.
Extraction of collagen ex vivo is generally achieved via incubation in acidic or enzymatic media, which results in the breakdown of natural crosslinks of collagen [29]. The resulting water-soluble material therefore presents minimal applicability in the physiological environment, so that synthetic routes aiming to restore covalent crosslinks are key [30].
Chemical crosslinking of type I collagen ex vivo has been widely adopted to equip resulting materials with water insolubility and mechanical competence in water [31], although the limited chemical accessibility of collagen triple helices raises challenges in the control and adjustment of macroscopic properties [32]. Recent synthetic approaches to accomplish controlled elasticity and degradation include crosslinked fibrillated type I collagen systems [ 33 ], UV-cured methacrylated collagen networks [ 34 , 35 ], collagen crosslinking with macromolecular segments [ 36 , 37 , 38 , 39 ], ionic co-networks [ 40 ], and nanostructured collagen-polyester composite [41]. While single-phase collagen materials proved to display at least 30 wt.% mass loss following 48 hours in collagenase medium [33,34], the fabrication of composite materials enabled structural customisation in the form of e.g. fibrous membrane [15] and double-layer sponge [42], whereby a mass loss of up to 30 wt.% was measured following 30 days in vitro [41]. These reports therefore indicate that current synthetic routes still only partially enable the fabrication of single-phase collagen-based membranes with GBR-compatible resorption rate (> 1 month), whilst the use of secondary polymeric phase, e.g. aliphatic polyesters, is critical to ensure long-lasting membrane stability in vivo, with the consequent safety concerns of degradation products.
In an effort to address this challenge, we have recently developed a family of telopeptidefree collagen (i.e. AC) systems functionalised with ethylenically unsaturated monomers, e.g. 4-vinylbenzyl chloride, that successfully generate hydrogel networks under light exposure with no addition of secondary crosslinker [ 43 , 44 ]. This strategy directly enabled customisation of properties according to the type of monomer that was chemically coupled to the collagen backbone [43, 45 , 46 ]. Additional biofunctionalities could be successfully introduced in a drug-free fashion by exploiting monomer-induced secondary interactions. For example, these molecular effects could generate drug-free inhibition of matrix metalloproteinases (MMPs) [44,47,48], ultimately delaying the enzymatic cleavage of the collagen network in vivo for durable material stability. The aim of this work was therefore to realise a single-phase and long-lasting AC-based hydrogel network with GBR applicability by leveraging the knowledge gained with the monomer-induced customisation of AC photonetworks. Departing from current design approaches of three-dimensional fibrous assemblies [16], hybrid synthetic/natural polymer formulations [41] and multi-layer composite structures [42], we hypothesised that building a covalent network of collagen triple helices with controlled crosslink density, capable of mediating drug-free MMP inhibition could enable the fabrication of a hydrogel-based GBR membrane with competitive elasticity, long-lasting stability in vivo and soft tissue barrier functionality. Our design strategy to deliver this was centred on the sequential functionalisation of AC triple helices with both 4VBC, as the MMPinhibiting monomer, and MA, as the highly reactive monomer, and to investigate the effect of the UV-curing solvent as an additional experimental space to adjust hydrogel compressibility, proteolytic degradability and swelling properties. The thorough physicochemical characterisation was pursued (i) to identify specific atelocollagen formulations generating GBR-compliant membrane microstructure and macroscopic properties (i.e. long-lasting stability, relatively low swelling and mechanical competence in biological environments); (ii) demonstrate in vitro the superior prototype proteolytic stability over Bio-Gide ® as one of the clinical gold standard membranes; and (iii) corroborate in vivo the direct effect played by the prototype properties on the GBR performance, aiming to de-risk future product development.
Ninhydrin 99% was ordered from Alfa-Aesar (Massachusetts, USA). Absolute ethanol and diethyl ether were purchased from VWR internationals. All other chemicals were purchased from Sigma-Aldrich unless specified.

Sequential functionalisation of AC
A two-step functionalisation route was developed to accomplish covalent coupling of both 4VBC and MA residues to the AC backbone. In the first step, 4VBC-functionalised AC was prepared as previously reported [43,44]. The solution of AC was diluted to a concentration of

(2,4,6)-Trinitrobenzenesulfonic acid (TNBS) and Ninhydrin assays
Both TNBS and Ninhydrin assays were used to measure the consumption of free AC's amino groups and respective degree of AC functionalisation, as described previously [41,42,45]. For the TNBS assay, the molar content of primary free amino groups and degree of functionalisation (F) were calculated via Equation 1 and Equation 2, respectively: where Abs (346nm) is the absorbance value recorded at 346 nm; 2 is the dilution factor; 0.02 is the volume of the sample solution (in litres); 1.46·10 4 is the molar absorption coefficient for 2,4,6-trinitrophenyl lysine (in M -1 cm -1 ); b is the cell path length (1 cm); x is the dry sample weight; and mol(Lys) AC and mol(Lys) funct . represent the total molar content of free amino groups in native and functionalised AC, respectively.
Confirmation of collagen functionalisation was obtained via Ninhydrin assay. 10 mg of the dry sample was mixed with 4 ml of distilled water and 1 ml of 8 wt.% Ninhydrin solution in acetone. The mixture was reacted at 100 ˚C for 15 mins, followed by cooling in ice and the addition of 1 ml of 50 vol.% ethanol in distilled water. The molar content of amino groups was measured by reading the absorbance at 570 nm against the blank. A standard calibration curve was prepared with collagen samples of known mass.
Three replicates were used for each assay, and data presented as mean ± SD.

Circular dichroism
The circular dichroism (CD) spectra of native and functionalised AC samples were acquired using a Chirascan CD spectrometer (Applied Photophysics Ltd) using 0. where MRW is the mean residue weight and equals to 91 g·mol -1 for amino acids [47];   is the observed ellipticity (degrees) at wavelength , d is the path length (1.0 mm), and c is the concentration (0.2 mg·ml -1 ).

Swelling, gel content and compression tests
Dry UV-cured samples of known mass (m d = 10-20 mg, n=4) were individually incubated in distilled water (1.5 ml) at room temperature. Following 24-hour incubation, water-equilibrated samples were collected, paper blotted and weighed (m s ). The swelling ratio (SR) was  Water-equilibrated hydrogel discs (Ø: 14 mm; h: 5-6 mm, n=3) were compressed at room temperature with a compression rate of 3 mm·min -1 (Instron ElectroPuls E3000) and a 250 N load cell. The dimensions of the hydrogel samples (Ø: 14 mm; h: 5-6 mm) were selected to minimise risks of sample tilting prior to or during the test, and to ensure uniaxial compression, in line with recent publications [36,43,49,66]. The compression modulus was quantified as the slope of the linear plot region at 25-45% compression in the stress-compression curves, at which range any potential risks of a platten effect are minimal.
Data of swelling ratio, gel content and compression modulus were presented as mean ± SD.

Scanning electron microscopy (SEM)
The morphology of UV-cured hydrogel surface and cross-section was explored via variable pressure SEM (Hitachi S-3400N VP, 60-70 Pa) combined with a Deben cool stage control (Model: LT3299). VP) combined with Deben cool stage control (Model: LT3299) was also employed to observe cell attachment after 7-day culture.

Subcutaneous stability test in vivo
All animal studies were conducted under procedures approved by the University of Leeds subcutaneous tissues were then bluntly dissected using artery forceps to create two pockets so that the PBS-swollen hydrogels were individually implanted within these pockets. The wounds were closed using 5-0 ethilon sutures. The wound was cleaned with sterile injection water and the triflurane switched off and the oxygen left on until the rats were fully recovered.
A buprenorphine (0.03 mg•ml -1 , 300 μl) injection was given to the rat. Following 4-week implantation, the rat was sacrificed using a carbon dioxide chamber for 6 minutes, followed by cervical dislocation. The samples and the surrounding tissue were then dissected out, photographed and fixed in 10% neutral buffered formalin (NBF).

In vivo GBR pilot study
A 30-day proof-of-concept study in vivo was carried out in a rat calvarial defect model using eight 3-month-old Sprague Dawley rats. The objectives of this study were to corroborate the results obtained in vitro and to build the foundation for a future larger animal study, derisking animal testing in the early stage of prototype development, in line with the principles of the 3Rs (Replacement, Reduction and Refinement). The experimental GBR procedure was performed as previously reported [50,51]. Two full-thickness calvarial bone defects (Ø: 5 mm) were created with a trephine. A sample of the commercial membrane (Bio-Gide ® , Geistlich) was trimmed to 6 mm diameter and always implanted to the intracranial side of the defect, as one of the leading GBR membrane gold standards. A commercial sample of deproteinised bovine bone mineral graft (Bio-Oss ® , Geistlich) was carefully applied to fill the bone defect area, whereby a negative control group without Bio-Oss ® was also used.
Subsequently, either the ethanol-disinfected hydrogel 4VBC-MA* (Ø: 8 mm, UV-cured in I2959-supplemented PBS solution) or Bio-Gide ® (as control, Ø: 8 mm) was applied to the extracranial side to assess the barrier functionality in the most degradative environment of the presented GBR model [14]. Finally, the flap was sutured in layers to close the wound.
The commercial membranes were always oriented with the rough surface towards the bone defect. Two rats of each group were sacrificed by schedule 1 method at both 1 and 4 weeks, and the samples were removed for histological analysis. The experimental groups are presented in Table 1.

Histology
The rat calvarium samples (including the materials) were fixed in 10% NBF. Following decalcification, the specimens were embedded in paraffin for sectioning. Anterior-posterior consecutive sections of 8-10 µm every 100 µm were cut with a fully motorised and programmable rotary microtome (Leica RM 2265) and stained with haematoxylin and eosin (H&E) according to the experimental groups presented in Table 1.

Statistical analysis
Statistical analysis was carried out using OriginPro 8.51. Statistical differences were determined by one-way ANOVA and the post hoc Tukey test. A p value less than 0.05 was considered to be significantly different. Data are presented as mean ± SD.

Results and Discussion
The fabrication of a GBR membrane was pursued via sequential functionalisation of AC with 4VBC and MA residues, followed by solubilisation in an I2959-supplemented aqueous solution and UV curing ( Figure 1). Coupling of both 4VBC and MA residues to lysine functions was hypothesised to generate a mechanically competent UV-cured material with enhanced enzymatic stability, reduced swelling ratio and barrier functionality, due to the nearly complete functionalisation and crosslinking of AC's primary amino groups, and the capability of 4VBC's aromatic residues to chelate and deactivate collagen-degrading MMPs in vivo [44]. The characterisation of the AC products will be presented at molecular, micro and macroscopic scales, and following L929 cell culture in vitro. Ultimately, a proof-ofconcept study in vivo in both a subcutaneous implantation model and a GBR model in rats will also be discussed to explore the material degradability and tissue formation following material implantation.

Chemical characterisation of sequentially functionalised AC
UV-cured collagen hydrogels have been shown to display adjusted macroscopic properties depending on the degree and type of covalent functionalisation of respective hydrogel-forming collagen precursors [30,46,52]. For example, an increase in the degree of methacrylation was demonstrated to directly correlate with the gel content (as an indirect measure of molecular crosslinking density), as well as the compression modulus and proteolytic stability (at the macroscopic scale) of respective UV-cured AC hydrogel [43].
Given the relevance of aforementioned macroscopic properties on GBR functionalities, functionalisation of AC was therefore pursued via sequential reaction with both 4VBC and MA ([monomer]·[Lys] -1 =25), aiming to accomplish nearly complete derivatisation of AC's lysines and exploit the relatively high reactivity of MA [47], as well as the molecular rigidity and metal-chelation capability of 4VBC [44]. Both TNBS and ninhydrin assays have been reported to be reliable methods to quantify the molar content of free amino groups [20,34,47] and were therefore selected to indirectly determine the degree of functionalisation in obtained AC products. Both assays confirmed a decreased molar content of free primary amino groups in 4VBC-reacted AC ([Lys]= 2.89 ± 0.06  2.38 ± 0.02 mol·g -1 ), corresponding to an averaged degree of functionalisation of 18 mol.% (Table 2), in line with previous reports [43,44].  Figure S1).
In contrast to previous reports [33,34,35,43], this sequential functionalisation strategy therefore proved successful to enable nearly-complete functionalisation of AC amino group terminations with two distinct monomers, promising a wider range of macroscopic and degradation profiles in the absence of dispersed [6,41] or crosslinked [37,39] synthetic phase.
Together with the confirmation of sequential functionalisation, the reaction product of AC was characterised by CD spectroscopy to elucidate the impact of sequential functionalisation on AC's triple helix organisation ( Figure 2). Dichroic patterns of sample 4VBC-MA proved to be comparable to the ones of native AC, whereby a negative peak at about 198 nm was observed corresponding to the presence of left-handed polyproline II helices; as well as a positive peak at about 220 nm, attributed to right-handed triple helices.

Figure 2.
Aforementioned CD spectra proved to be in stark contrast with the CD spectrum of gelatin [45,46], which shows no positive CD peak and confirming the presence of a randomly oriented polypeptide configuration. Other than the CD patterns, the magnitude ratio between positive and negative peak intensities (RPN) was also calculated and found to be equal to 0.12 and 0.088 in the case of native AC and SAP, respectively. While the former value appears to be in line with previous reports on type I collagen [30,43,45,46], the decrease of RPN in the SAP suggests a reduction in the content of AC triple helices. This observation is likely due to the nearly complete derivatisation of triple helix-stabilising primary amino groups of lysine terminations [47,53] and to the partial introduction of bulky 4VBC aromatic groups (Table 2).

Fabrication of UV-cured hydrogels
When 4VBC-MA was dissolved in I2959-supplemented aqueous solutions, full gelation was accomplished following exposure to UV light, an observation that supported the synthesis of a covalently crosslinked network of sequentially functionalised AC triple helices.
No detectable macroscopic differences were observed between hydrogels 4VBC-MA* and 4VBC* ( Figure S2, Supp. Inf.), and with respect to state-of-the-art glutaraldehyde crosslinked collagen [54]. Cool-stage SEM of hydrogels 4VBC-MA* revealed a homogeneous distribution of interconnected micropores (Ø ≤10 μm) across the material (Figure 3). High pore interconnectivity has been observed in previously reported UV-cured networks of covalently crosslinked collagen molecules [30,33,35,46], and indirectly suggests densification of collagen molecules via covalent crosslinking as well as improved degradability and mechanical properties [36]. Furthermore, control of internal pore size can also be leveraged to minimise risks of soft tissue cell infiltration in GBR membranes, as  Table 2). These results therefore confirm the direct effect of this molecular parameter on the macroscopic properties of respective UV-cured hydrogels, as previously observed with single functionalised collagen precursors [43,46,51]. whereby the value of G' (8149 ± 345 Pa) was found to be more than 200 times higher than the value of G'' (40 ± 12 Pa) following UV treatment ( Figure 4A). The hydrogels 4VBC-MA* therefore revealed a 20-fold increase in storage modulus compared to hydrogel 4VBC*, i.e.
obtained via UV-curing AC precursors bearing 4VBC residues only [43]. These observations therefore demonstrate the significantly increased elasticity of UV-cured sequentially functionalised AC hydrogels, which is key to enable hydrogel applicability in GBR therapy.
The aforementioned values of storage modulus measured in hydrogels 4VBC-MA* proved to be higher than the ones reported with both orthogonally crosslinked collagen-hyaluronic acid co-networks [37] and carboxymethyl cellulose-crosslinked collagen [40], and close to the ones of nanostructured collagen-PLGA composites [41], whilst higher values were revealed when type I collagen hydrogels were crosslinked with high molecular weight dextran [36].
These results therefore indicate the significant effect of the presented sequential functionalisation route on the mechanical properties of collagen-based materials prepared in the absence of either secondary polymers or collagen self-assembling step.
Other than the rheological differences at equilibrium, the solution of sample 4VBC-MA in Pa; G''= 5 ± 1 Pa) ( Figure 4B and C).
As a well-known ampholyte, environmental factors, such as pH, ionic strength and salt concentration, as well as the amino acidic composition, affect secondary interactions and long-range configuration of collagen molecules [34,40,58 ]. For instance, incubation of crosslinked type I collagen in aqueous solutions of decreased acidity was exploited to accomplish hydrogels with decreased swelling by controlling the electrostatic interactions between amino acidic side chains [59]. In this study, the sequential functionalisation with both 4VBC and MA residues is expected to contribute new secondary interactions between AC molecules. This observation is supported given the pH-dependent capability of aforementioned aromatic and methacrylate groups to mediate - stacking interactions and hydrogen bonds, respectively, the nearly complete consumption of AC's free amino groups and the consequent decrease in AC's isoelectric point. Indeed, the isoelectric point of collagen was found to decrease from ~6.5 to ~5.5 following functionalisation of 30 mol.% of lysines with methacrylate residues [34], in agreement with the increased molar content of negatively charged carboxylic functions in the collagen backbone. Therefore, the increased molar content of MA (compared to 4VBC) residues and the decreased isoelectric point in the SAP are attributed to explain the above-mentioned decrease of storage and loss moduli in AC networks prepared in solutions of increased pH. With the increase in pH, the presence of OHions generates weaker hydrogen bonds [60] as well as electrostatic repulsion between AC side chains terminating with deprotonated carboxylic terminations [34], resulting in decreased shear moduli, as observed with UV-cured MA-functionalised AC [43]. Unlike the case where UV curing was carried out at increased pH (Figure 4 B-C), significant secondary interactions could be detected when the sample 4VBC-MA was solubilised in the 10 mM HCl solution (pH ~ 2), whereby no cross-over point and a predominantly elastic response was measured following activation of UV light ( Figure 4A). Given that water acts as a plasticiser in biopolymers [32], it is critical that respective water-induced swelling does not deteriorate the clinical handleability of the collagen membrane and that the membrane dimensional conformation with the tissue defect is ensured. Indeed, a relatively low swelling ratio in the GBR membrane is important for clinicians to ensure primary wound closure. Conversely, an unexpected /uncontrolled swelling ratio during the early healing phase might lead to membrane /graft exposure and healing by secondary intention, which would compromise the regeneration outcome.

Physical characterisation: swelling ratio, gel content and compression properties
Consequently, it was important to measure the swelling behaviour of accomplished UVcured hydrogel networks. All dry collagen samples took up, and swelled when incubated in, distilled water, whereby a swelling ratio (SR) in the range of 350±48-684±76 wt.% was measured upon solvent equilibration ( Figure 5). Especially the SR of the PBS-cured hydrogels was found to be comparable to the one of Bio-Gide ® (SR= 289±22 wt.%, p= 0.0602) and significantly smaller than the one measured in crosslinked collagen systems intended for GBR therapy [38,42,63] as well as UV-cured homo-networks of either 4VBC-or MA-functionalised collagen [43,44,46]. From a physical property point of view, the aforementioned swelling behaviour therefore support the GBR applicability of this hydrogel, aiming to reduce the volumetric expansion of the membrane at the defect site in vivo following contact with the physiological medium and to ensure tissue occlusivity [14,49]. The aforementioned relatively low values of SR can be attributed to the significant crosslink density in the resulting UV-cured network and the relatively high concentration of collagen used in the UV-curing aqueous system. Given the large swelling ratio in physiological conditions and the fact that water acts as a plasticiser for collagen, the attention moved to the characterisation of the mechanical properties in the water-equilibrated collagen networks. Samples with a diameter/height ratio of ~2 were prepared to minimise risks of sample tilting during compression, which is typically observed when hydrogel samples with a relatively large height are measured. All samples described a J-shaped stress-compression curve ( Figure 6A), which is typical of biological tissues [36,43,49,66] and which is attributed to the complex interplay of aqueous fluid and covalently-crosslinked collagen molecules under compression [68]. The compression properties of the AC networks varied according to the UV-curing solvent, whereby the PBS-cured samples displayed the largest compressibility (up to nearly 90% compression) and significantly reduced compression modulus (E c = 22±9 kPa), compared to both acid-cured samples (E c = 155±24-178±20 kPa) ( Figure 6B). This variation in compression modulus agrees with previously discussed trends in shear moduli (Figure 4), confirming the suitability of selected sample geometry and compression setup, so that a multimodal mechanical characterisation was accomplished. The variation in compression modulus also reflects the increased compressibility and decreased gel content measured in AC networks UV-cured in PBS ( Figure 5). The increased compressibility presented by the PBS-cured hydrogel networks is key aiming to accomplish safe membrane implantation and fixation at the critical-sized defect site, minimising risks of membrane collapse in the defect and /or membrane damage, including curling or wrinkling observed following contact with body fluid [16].

Proteolytic stability in vitro and subcutaneous implantation in vivo
Samples UV-cured in PBS were subsequently selected for degradation tests in collagenase media, given their relatively low swelling ratio, relatively high compressibility and acid-free synthesis. These three features were considered key for GBR therapy, aiming to ensure occlusivity, elasticity and surgical handling, as well as cell tolerability and safety in vivo, respectively. Following 14-day enzymatic incubation (37 ºC, 1 CDU•ml -1 ), samples 4VBC-MA* successfully displayed significantly higher relative mass at both days 4 and 14 ( rel = 73±8 wt.%  50±4 wt.%), with respect to both controls of UV-cured 4VBCfunctionalised homo-network ( rel = 46±2  24±4 wt.%) and the commercial collagen membrane Bio-Gide ® ( rel = 19±5  14±4 wt%) ( Figure 7A). The enhanced proteolytic stability of the UV-cured samples 4VBC-MA* was further confirmed in solutions supplemented with increased collagenase concentration (2.5 CDU•ml -1 ), whereby nearly 50 wt.% of the original mass was still retained ( rel = 57±2  47±3 wt.%), whilst almost all sample of Bio-Gide ® was lost ( rel =2±4  1±2 wt.%) ( Figure 7B). These gravimetric results were in agreement with the digital macrographs captured during sample incubation in vitro, whereby the hydrogel 4VBC-MA* revealed minimal changes in macroscopic integrity in contrast to the samples of the collagen membrane ( Figure 7C). The aforementioned degradation trends were also confirmed when control samples of native AC were used, which revealed a 10-fold decrease in relative mass with respect to the UV-cured samples 4VBC-MA* following 4-day incubation with increased collagenase concentration (2.5  5 CDU ml -1 , Figure S4, Supp. Inf.). Overall, the presence of 4VBC-crosslinked AC molecules is expected to induce cation- interactions between 4VBC's aromatic rings and active zinc sites of collagenases, so that drug-free enzymatic deactivation can be successfully accomplished with minimal peptide bond scission [43]. Together with 4VBC, the presence of MA in the SAP is on the other hand beneficial to accomplish increased crosslink density following UV curing and delayed AC degradation. The 14-day proteolytic stability (μ rel = 47±3 wt.%) of 4VBC-MA* in vitro was significantly higher than that reported for hyaluronic acid-collagen co-networks [37], UVcured methacrylated collagen [34], as well as PVA- [66], genipin- [67] and PEG-crosslinked collagen [ 69 ], whilst, as expected, it was lower than that of carboxymethyl cellulose-crosslinked collagen [42], in light of the protease insensitivity of cellulose.

Biocompatibility and cell proliferation in vitro
Consequent to the degradation tests, efforts focused on assessing the tolerability of the accomplished AC hydrogel 4VBC-MA* with L929 fibroblasts, aiming to confirm cell viability, the absence of any hydrogel-induced toxic effects, and cell barrier functionality (Figure 8).
Accordingly, dry UV-cured samples were incubated in a 70 vol.% ethanol solution under UV exposure, to achieve material disinfection [70,71]. Live/dead staining of the L929 fibroblasts    (Figure 7). These observations speak in favour of a surface erosion rather than bulk degradation mechanism, in agreement with previous degradation studies on collagen-based hydrogels [52,73]. According to a surface erosion mechanism, larger pores are predominantly formed in the outer layer rather than on the hydrogel core, generating minimal risks on the long-term barrier and tissue integration membrane functionalities.
The aforementioned data therefore demonstrate that the presented sequential collagen functionalisation route enables the simple fabrication of a biocompatible hydrogel with convenient microstructure for localised fibroblast growth and barrier functionality, yet avoiding complex manufacturing steps and multi-layered membrane architectures [15,16,19,42].

In vivo pilot study of GBR functionality
Having confirmed its biocompatibility and barrier functionality in vitro, as well as its 4-week material integrity in vivo, the attention focused on exploring the performance of the hydrogel as a GBR barrier membrane in a GBR animal model. Given the unknown tolerance in vivo of this previously-unreported AC membrane, a proof-of-concept study was carried out in rats using a relatively low number of animals, in line with the principles of the 3Rs for animal testing and aiming to lay down the research foundation for a future larger investigation in vivo. Samples of the AC hydrogel and Bio-Gide ® were employed as the experimental group and the clinical gold standard, respectively. Calvarial defect models are often employed to assess the in vivo bone regeneration capacity, whereby a critical size defect is created in the exposed calvarial bone, treated with the experimental sample, followed by repositioning and suturing of the soft tissue [6,17,23,50]. Although this model offers a relatively easy surgical procedure to assess the material-induced bone regeneration, risks persist with respect to soft tissue cell infiltration from the underlying dura mater, especially when the membrane is not completely conformed to the surgical site. To minimise risks of soft tissue infiltration, a barrier GBR defect model was created in the calvaria of rats [ 74 ]. To assess the barrier functionality in the most degradative environment of the presented GBR defect [14], samples of 4VBC-MA* were always applied to the extracranial side ( Figure 10A), i.e. facing the periosteum and soft tissue, whereby control groups were treated with Bio-Gide ® . Compatibly with the nature of the proof-of-concept study, the intracranial side, i.e. facing the dura mater ( Figure 10A), was on the other hand always covered with samples of Bio-Gide ® . As one of the leading GBR membrane gold standards, Bio-Gide ® has indeed been extensively documented in the literature to prevent the ingrowth of cells coming from the dura mater [75,76,77]. Ultimately, the effect of the graft material Bio-Oss ® in the critical size defect was also explored. The space between the two defect sides was still observed, whereby the formation of densely packed tissue was detected within the bone defect area and around the Bio-Oss ® particles ( Figure 10D-E & H-I, see black arrows). The distance between the two membranes proved to be decreased in the blank defects ( Figure 10B-G) compared to defects filled with Bio-Oss ® ( Figure 10D-I), suggesting that the application of the membranes alone is insufficient to withstand the mechanical loads and preserve the integrity of the defect gap.
Increased cell density was also observed in the graft-filled defects ( Figure 10I, see black arrows), likely due to the fact that Bio-Oss ® promotes osteoblast migration from the edges, towards the centre, of the defect, in line with the osteoconductive functionality of the selected graft material. Nevertheless, implantation of the membranes for one week in vivo may be too short for visualising any de novo bone tissue formation [78], as also supported by the presence of small gaps at the membrane-defect interface ( Figure 10B-D & F-I). This observation is in line with the initial inflammation phase of the bone regeneration process [16], prior to the subsequent occurrence of angiogenesis, granulation tissue, and remodelling [79].
After four weeks of implantation, both samples of Bio-Gide ® (Figure 11A-D) and hydrogel 4VBC-MA* ( Figure 11E-H) remained intact, supporting the long-lasting proteolytic stability of the AC hydrogel previously observed in vitro and in vivo (Figure 7). Overall, these preliminary in vivo results support the integrity and cell /tissue tolerability of the hydrogel following four-week implantation in vivo, as observed in the case of Bio-Gide ® , and in line with enzymatic degradation (Figure 7) and barrier functionality ( Figure 8) results obtained in vitro. Further larger scale follow on study should be carried out in vivo to assess hydrogel impact on bone regeneration and longer-term hydrogel stability in the GBR defect model, which can take advantage of more advanced 3D imaging technologies, such as micro-CT.

Conclusions
The sequential functionalisation of AC with two distinct ethylenically unsaturated monomers, i.e. 4VBC and MA, successfully enabled the fabrication of a UV-cured hydrogel network with competitive proteolytic stability, compressibility and swelling ratios with respect to the corresponding homo-networks, as well as increased proteolytic stability with respect to Bio-Gide ® , as one of the current GBR collagen membrane gold standards. The selection of acidic or basic UV-curing aqueous systems proved to affect gelation kinetics, swelling ratio and compression properties, due to the effect of ionisable terminations of AC, covalently coupled monomers and the complete consumption of primary amino groups. As observed in collagenase-supplemented media in vitro, the UV-cured sequentially functionalised samples proved to remain intact for at least four weeks following subcutaneous implantation in rats, and to ensure space maintenance in a proof-of-concept study in a GBR model in rats. These in vivo results therefore provide compelling evidence aiming to de-risk future prototype development and support the additional large scale preclinical testing of this long-lasting AC membrane, following the principles of the 3Rs for experimental animal studies. A follow-on larger scale investigation in vivo will be key to confirm the longer-term stability of this material and its impact on bone regeneration, prior to moving forward with a first-in-human clinical evaluation.