Microstructure Reserved Bacterial Nanocellulose Functional Composite And Its Crucial Prepareion Technology

Bacterail nanocellulose(BNC)is a microbiologicaly produced cellulosic hydrogel.It possesses incredible potential in biomedical applications due to its extracellular matrix(ECM)like natural nanofibrous microstructure and versatile decent properties(such as high hydrophilicity and biocompatibility).These widespread applications include surgical dressings,artificial blood vessels,and cartilage substitutes.However,characteristics of pristine BNC materials cannot meet all the requirements of specific medical applications.First,insufficient tensile strength and suture retention strength in a highly swollen state largely restricted its applications in implantable devices or closuring dressings with suture demands.Second,no antibacterial capacity of BNC cannot satisfy general anti-infective requirements for most medical applications.Third,BNC can not rehydrate to hydrogel state after conventional drying as general polymer hydrogels do,which also greatly limites its applications.Besides the performance problems,as a microbial product,inefficient production with tranditional static fematations also restrictes its commercialization applications.BNC hydrogels are valued for the innate ECM-similar nanofibrous structure,which is formed in microbial fabrication process and is still difficult to be obtained via a bionic manufacture.However,tranditional compositing methods would inevitably disrupte the nano-fiber 3D network structure and its corresponding features,which seriously affectes follow-up implementing effects.Thus,conservation and utilization of the unique innate microsturcture of BNC has become a hot research field in BNC based fuctional materials.This doctoral thesis carried out the investigations in three kinds of microstructure-reserved BNC fuctional composites and its preparations,which aimed at remedying the four performance defects of innate BNC material(namely,strength reinforcement,production efficiency improvement,antibacterial functionalization,and spontaneous structural recovery after dehydration).It has been concluded as follows:Fistly,this study proposed an in situ dynamic biofilm culture technology to create a fabric-frame reinforced nanocomposite of BNC hydrogel with superior strength,but retained BNC native attributes,in a self-designed horizontal rotating bioreactor.In the bioreactor,BNC was spontaneously immobilized on a semi-submerged roller and grew thick through a "bio-coating" process.Both the biofabrication process and the properites of the fabric-reinforced BNC composites were investigated.In the study on biofabrication process,the fermentation process of tranditional static cultures was compared with our dynamic biofilm cultures.By using the proposed technology,production time could be reduced from 10 days to 3 days to obtain a desirable hydrogel sheet with approximately the same thickness in static cultures.In the static cultures,substrate mass transfer through a growing cellulose layer into an active cell zone is a crucial limiting factor.However,in this bioreactor,the substrates(mainly glucose and oxygen)initiatively replenish the surface of product cellulose in a rotational order,which has more advantages in substrate mass transfer than in static cultures,which greatly improved its productive rate and total BNC yeilds rather than cell concentretion.In this research,the rotating speed was optimized basd on the computational analysis of effective substrate flux into the active cell zone.By utilizing the second Fike's law,a reduced mathematic model was proposed to calculate substrate flux into cellulose surface with logical suppose and predigestion.And a fluorescent staining method was utilized to distinguish the active cell zone and two different substrates were studied.The penetrating distance of substrate flux into cellulose layer was found depending on the rotating speed and the diffusion coefficient of substrate in BNC matrix,and independent of substrate concentration in culture media.A too short penetrating distance would make the active cell zone insufficiently supplied,while a t oo long penetrating distance would lead to insufficient utilization of substrate flux.To our knowledge,it is the first study to explore the possible mechanism of substrate mass transfer influencing BNC production in rotating mode.This bioreactor and methods are suitable for scale-up and efficient industrial production.In the aspect of fabric reinforced BNC materials,cotton gauzes(CG),a commercial wound dressing textile,was used as a fabric skeleton to fabricate a CG-reinforced BNC hydrogel(BNC-CG),which had superior strength but retained BNC native attributes.The mechanical properties(tensile strength,suture retention strength)and gel characteristics(water holding,absorption and wicking ability)of the fabric-reinforced BNC hydrogel were investigated and compared with those of ordinary BNC hydrogel sheets from 10-day cultivation.The results domestrated that the BNC-CGs had all impotant BNC hydrogel attributes,such as high water content,water absorption,water-holding capacity and the typical nano-fibrillar sturcture,although there was slight difference in the water content and liquid wicking transport betwenn the BNC hydrogels from the dynamic biofilm cultures and those from static cultures.The CG skeleton strengthened the BNC hydrogel body by two orders of magnitudes,and the CG itself was greatly improved by surface modification with nanocellulose.This work showed that fabric reinforcement could improve the operational reliability of BNC-based hydrogels.And in another perspective,coating BNC hydrogle on CG is a upgrading of conventional gauze to a material with desired properties.Better than BNC or CG separately,the BNC-CGs was highly interesting for future studies regarding advanced wound dressings.Secondly,in situ biosynthesis method was regarded as a feasible compositing method with minor modifications in the innate microsturcture of BNC,which incoporated other polymers into the BNC nano-fibers through dissolving the cellulose-compatible polymers in the culture media.In order to improve the antibacterial ability,chitosan(CS)was composited with BNC by using the in situ biosynthesis of static cultivations.The influence of acid type,CS concentration,p H value and autoclaving procedure on both solubility of CS in culture media and biofabrication of CS/BNC composites were investigated.The results indicated that acetic acid aqueous solutions were the optimal media for CS/BNC composites biosynthesis.To mixe CS with culture media firstly and autoclave synchronously gave better results as compared to those separately autoclaved after separate preparation of CS aqueous solution and culture media.With the acetic acid-CS contained media,CS was incorporated into BNC nanofiber successfully and existed stably in nanofibers with CS content of 35-48%.However,because of the antibacterial effects of CS on the production strain(Gluconacetobacter xylinus),the CS/BNC hydrogel from static cultures showed much sparse networks as compared to the prinstine BNC hydrogel.And the CS/BNC composite hydrogel was too fragile and thin to accomplish mechanical performance evaluations.Thus the CS/BNC hydrogel obtained from static in situ biosynthesis was less possible for practical use.In order to improve the performance of the CS/BNC composite hydrogel,an in situ biosynthesis technology was proposed by using dynamic biofilm cultures in a rotating drum bioreactor,where the drum was composed of CG.CS was dissolved with acetic acid to very low concentration of 0.25-0.75 w/v% in the culture media.It was found that CS finally occupied 40-44% of CS/BNC dry weight.FE-SEM observation confirmed conservation of the nano-fibrillar networks of BNC,similar to that of prinstine BNC,and found CS incoperated in the nanofibers.ATR-FTIR showed a decrease in the degree of intra-molecular hydrogen bonding.Water absorption capacity was improved after compositing with CS.The fabric-reinforced CS/BNC composite exhibited bacteriostatic properties against Escherichia coli and Staphylococcus aureus.Thus,the CG reinforced CS/BNC composites simultaneously possess an ECM-similar nanofibrous structure,hydrogel attributes of prinstine BNC,CG-skeleton reinforcements,and bacteriostatic properties of CS,which would facilitate more widespread applications of BNC composites in the biomedical materials area.Finally,a concept of free standing hydrogel film with high degree one-dimensional swelling seems very promising for a wide range application,which is still far from realization.BNC is a naturally occurring hydrogel,composed of multi-layered nano-fibrous networks,and exhibits a strict one-dimensional shrink property after conventional dehydration to form a sturdy ultra-thin film.However,it is incapable in rehydration and swelling again due to massive H-bondings between cellulosic nanofibers,which is regarded as its main critical defect.But it provides us a glimmer of feasibility to fabricate a novel free-standing nanofibrillar hydrogel film that possesses high degree one-dimensional swelling ability by overcoming the restriction of re-swelling difficulty.In this study,several polyelectrolyte(PE)polymers were penetrated into the nanofiber network of pristine BNC and then dehydrated into a PE/BNC composite thin film.This new sort of free-standing thin film was capable of realizing the following attractive attributes,including perfect one-dimensional swelling,p H-controlled switch-on,nano-fibrillar structure and high volumetric swelling degree,which was able to swell up in thickness of around 100-fold without any planar size deformations.To verify the proposed hypothesis,three different PE/BNC films were prepared as examples with three different PEs consisted of similar main chain but different electriferous groups,i.e.chitonsan(CS)with cationic amino,carboxymethylcellulose(CMC)with anionic carboxyl,and carboxymethyl chitosan(CMCS)with both the two groups,respectively.They were verified with discrepant swelling-enabled p H range with each others.A two-chamber diffusion experiment revealed that the film has a good p H-selective molecule permeability which behaves as zero-order release in swelling state when compared to a rapid release in non-swelling state.MTT assay results showed that the all-natural polymer composed films displayed good cytocompatibility.This kind of BNC based composite films was only a small number of a self-supporting film with large-degree 1D swelling so far,and was the only one to have a th ickness of sub-100 ?m and linear swelling ratio of large than 100-fold.It could serve a burgeoning platform for multitudinous relative devices including active component of biosensor,gating valve,drug delivery,and microfluidic actuator.