The Establishment Of Foreign Body Reaction Induced Prevascularized Chamber And Its Promotion Of Hair Regeneration

BackgroundCell therapy is the fourth and final therapeutic pillar of global healthcare.It is critical for the development of regenerative medicine,which aims to replace or regenerate cells,tissues or organs in order to restore or establish normal function.For over 20 years,the cell-based therapy has successfully manufactured products and cured patients.However,few tissues are capable of regeneration.Hair follicles are abundant micro-organs in the human body.Their morphogenesis has been controlled by the interaction of epithelial and dermal components,with a rhythmic cyclical change.It is currently a hot research field in regenerative medicine.Exploration in hair regeneration will bring significant reference to organogenesis in other systems,and also help to make therapeutic strategy for hair loss.Hair follicle reconstitution in vitro has not yet been successful due to the loss or reduction of signaling factors and cytokines fails to mimic hair follicle morphogenesis in vivo.Although several in vivo hair follicle reconstitution protocols(e.g.,silicone chamber assay,the patch assay)are available,they have several deficiencies.For example,the early phase of grafting in patch assay is associated with extensive apoptosis,which may be due to an inadequate blood supply,and result in low graft uptake.Besides,the formation of a single hair follicle requires many cells,especially when using the chamber assay.Therefore,further research is needed to generate a better strategy in improving the regeneration efficiency.Researches on the development of alternative hair regeneration strategy and tissue engineering suggest that an ideal transplant site for graft should(i)provide adequate tissue volume for graft survival,(ii)rapidly construct functional anastomosis between host and graft vasculature,(iii)facilitate dynamic communication between the graft and host circulation,and(iv)has low mechanical properties.Among these essentials,sufficient vascularization has been a most challenge.Classical vascularization approaches focus on the stimulation of vascular ingrowth into tissue constructs.However,these approaches face the problem that the average growth rate of newly developing microvessels is only?5 um/h,thus the complete vascularization needs a prolonged time period,which may lead to major tissue loss due to hypoxic conditions.As a result,"prevascularization" has emerged as a promising concept to generate a performed microvasculature in tissue conducts prior to their implantation.It will shorten the period during which the constructs suffer avascular and hypoxia conditions,and contribute to early phase survival and long term function of implants.However,a well-vascularized skin site will also suffer from the problems of low tissue volume,deficient metabolic exchange and robust mechanical properties.Substantial efforts need to be made to address these situations.Inspired by the innovative approach for islet transplantation in a foreign-body reaction(FBR)-induced subcutaneous prevascularized transplant pocket,we found that in-situ FBR could create a transplant site with large tissue volume and sufficient vascular networks.However,the mechanical properties and permeability of the prevascularized site were not investigated.Besides,excessive production and packaging of collagen fibers might hinder the successful transplantation.Thus,the timing of implantation is of great importance in determining the regeneration efficiency.Therefore,further studies are needed to fully harness the FBR process in a controlled manner so as to create a transplant site with a large tissue volume,well-vascularized microenvironment,highly permeable and low mechanical properties.Herein,we manufactured a modified transplant site,the prevascularized collagen fibers(PVCF)matrix,by temporary implantation of a medically approved catheter on the dorsum of nude mice.Specifically,different time points were set upon implant withdrawal to investigate the histological and mechanical assessments.Subsequently,the isolated trichogenic cells were transplanted into this matrix after implant withdrawal to regenerate hairs.Objective:(1)To prepare a prevascularized collagen fiber(PVCF)matrix via controlling the duration of foreign body reaction(FBR),and evaluate the evolution of physiological properties;(2)To explore the mechanical properties of the optimally preprogrammed PVCF matrix;(3)To investigate the function of the optimally preprogrammed PVCF matrix as a modified transplant site in improving the hair regeneration efficiency.Methods:Conduction of FBR-induced PVCF matrix and its physiological evaluationMedical approved sterilized indwelling silicone catheter(China)with a length of 20mm and a diameter of 2.5mm and silicone caps(Dow Corning,Midland,MI,USA)with a diameter of 8mm were sterilized prepared to create in-situ FBR-induced PVCF matrix on adult athymic nude mice.A 5 mm lateral vertical incision was made caudal to the dorsum with scissors and a small entrance to the subcutaneous gap was created using blunt dissection.The catheter was implanted into the space parallel to the midline incision.The incision was closed with a 4-0 suture.After implantation,blood proteins adhered to the catheter,resulting in the formation of densely vascularized tissue,which was clearly visible after histological processing.The removal of the catheter prior to transplantation revealed a vascularized chambers allowing for cell transplantation.After 1 week,2 weeks,3 weeks,4 weeks and 5 weeks of subcutaneous implantation,the FBR was terminated respectively in order to investigate the influence of FBR duration on morphological properties.Light microscope,scanning electron microscope,HE staining,Masson staining,immunohistochemistry and immunofluorescence staining was carried out to identify the properties.Evaluation of the mechanical properties of the FBR-induced PVCF matrixAll rheology oscillation experiments were performed on a TA Discovery Hybrid Rheometer using 8 mm parallel-plate geometry at 25?.During oscillation-frequency experiments,the samples of PVCF matrix and skin sample were endured the shearing rate ramping from 0.1rad/s to 100rad/s under a constant strain of 0.5%.The viscosity measurements were conducted from 0.1 to 100 s-1 at a fixed strain of 0.5%.For tensile tests,cuboid sample(30mm height,1mm depth,7?10 mm length)of PVCF matrix and normal skin were prepared and stretched at a speed of 20 mm/min to the breaking points on a universal tensile tester(Instron 5965).The tensile modulus was calculated as the slope in the linear region from the stress-strain curves.The viscoelastic property of these samples was measured by cyclic tensile tests and stress-relaxation test.For cyclic tensile tests,the matrix sample as well as the skin×sample bear a tensile strain of up to 33.3%and then released to 0%at the constant rate of 20mm/min for 10 cycles respectively.Another cyclic tensile tests with a higher strain of 66.7%is conducted on PVCF and a second run follows after 1h.Energy dissipation of PVCF matrix and normal skin in the first tensile cycle were calculated according to the area within the hysteresis loop of respectively.For the stress-relaxation test,each sample was stretched up to 66.7%strain at a constant rate of 20mm/min,and then was kept in the strained condition for 60 seconds.The reaction force to the constant strain was measured as a function of relaxation time.The variation between the reaction force at each instant time,F(t),and the reaction force at the beginning of stress relaxation,F(t0),was calculated.Since this samples came from different materials,this variation was normalized by dividing it by F(t0)corresponding to each sample.Transplantation of neonatal mouse skin cells into PVCF matrix to improve the efficiency of hair regenerationMouse epidermal cells(MECs)and mouse dermal cells(MDCs)were isolated from 0-2 days newborn C57BL/6J mouse skin.Briefly,the full thickness skin was removed and incubated in phosphate-buffered saline(PBS)(Gibco,Grand Island,NY,USA)with 0.1%(wt/vol)dispase(Sigma,St.Louis,MO,USA)at 4? overnight.After being rinsed 3 times in PBS,the skin was then split into epidermis and dermis with forceps.The dermis sheets were minced and digested in 0.2%collagenase(Sigma,St.Louis,MO,USA)at 37? for 1h.The epidermal sheets were minced and then incubated in 0.25%(wt/vol)trypsin(Invitrogen,Carlsbad,CA,USA)at 37?for 10 min with gentle shaking..After digestion,each component was filtered sequentially through 100 and 40 pm mesh cell strainers.Cells were collected by centrifugation at 320 × g for 5 min and resuspended in Dulbecco's modified Eagle medium(DMEM)(Gibco,Grand Island,NY,USA)for cell grafting.As for transplantation into the cylinder-shaped hollow PVCF matrix subcutaneously,DMEM suspensions of 20 uL with 1 × 106 MDCs and 0.5 × 106 MECs were prepared and pelleted by centrifugation.Nude mice were anesthetized by an intraperitoneal injection of 0.3ml pentobarbital sodium(0.3 g/100 ml),and the dorsum area of the sedated mice was cleaned with Betadine twice.Cranial to the superior edge of the implanted catheter,a 5 mm incision was made to gain access to the catheter.After different time points of FBR process,a 4 mm incision was made to gain access to the catheter,the tissue matrix surrounding the superior margin of the catheter was dissected to withdraw and remove the catheter alone without pulling out the surrounding prevascularized tissue.The cell suspensions were delivered into these remaining vascularized lumen of different groups respectively.Every individual incision was closed with a surgical suture.Animals from control group were grafted with the same amount of cell suspensions using the patch assay,which was performed as previously described.As for transplantation onto the flat PVCF matrix on the dorsal side of the mice,DMEM suspensions of 50 uL with 5 × 106 MDCs and 5 × 106 MECs were prepared and pelleted by centrifugation.At each different time points of FBR process,a 2 mm incision vertically to the covered rim was made on the sedated mice to remove the implanted cap easily without pulling out the surrounding tissues,yielding a flat,shallow pool-shaped and well-vascularized collagen tissue.The cell suspensions were then delivered onto these FBR-induced flat vascularized collagen matrix of different groups.the round opening was covered with a same-sized plastic film from preventing pollution by 6-0 suture,and the film was removed 1 week after transplantation.Animals from control group were grafted with the same amount of cell suspensions using the silicone chamber assay,which was performed as previously described.Subcutaneous transplanted sites were harvested 12 days after transplantation and processed for histological analysis.First,for gross appearance,hairs grown at graft sites were observed by light microscopy,photographed,stripped with forceps and spread on paper for counting.For histological analysis,the harvested transplanted sites were fixed in 10%(vol/vol)formalin overnight.After paraffin embedding,the tissues were processed for hematoxylin and eosin staining.To identify neovascularization by endothelial cells,an anti-CD31 antibody(Abcam,Cambridge,UK)was used for immunofluorescent staining.ResultsConduction of FBR-induced PVCF matrix and its physiological evaluationStereomicroscopy and SEM were used to observe the surface structural characteristics of the prevascularized capsule.By stereomicroscopy,the outer surface of the chamber was enclosed by a thin layer of fibrous tissue that was interspersed with dense neovessels.By SEM,the inner surface of the chamber was covered with cross-linked collagen fibrils that formed a porous mesh structure with tunable pore size from 0.1 to 10 ?m.Red blood cells and activated platelet were found within the collagen fibers network,together resembling a well-vascularized ECM.To further investigate the prevascularized chamber,the specimens were embedded in paraffin wax and stained with Masson's trichrome and immunohistochemistry for F4/80.By 1 week,newly formed thinning collagen fibers were observed lining the inner surface of the cylinder matrix,along with large amounts of F4/80 staining positive macrophages.By 3 weeks,well-organized collagen fibers were gradually deposited and getting thicker,accompanied by obviously presented newly formed blood vessels and inflammatory cells infiltration.With the progression of FBR continued,newly formed collagen fibrous transformed from well-organized fibers to dense and solid bundles,which was revealed by the formation of hypocellular avascular dense fibrous matrix at week 5.Specifically,the thickness of the collagen fibers throughout the whole progression was climbing and highest at week 3 and shrinked by week 5.Instead,neovascularization embedded among the collagen fibers was noticed from week 1(highest at 2 to 3 weeks and diminished afterwards).Inflammatory cells were evenly distributed along the collagen fibers,the number of nuclei peaked at 2 weeks after the implantation of the silicone catheter,indicating a reduction in inflammatory infiltration since then.Particularly,among the inflammatory infiltrating cells,the number of macrophages was highest at week 1,and decreased rapidly afterwards.Evaluation of the mechanical properties of the FBR-induced PVCF matrixThe viscosity of PVCF decreased steadily with the frequency increasing from 0.1 to 100 rad/s,exhibiting the typical shear-thinning behavior for this tissue.Additionally,compared with the normal skin tissue,the viscosity of PVCF was much lower and dropped more sharply in high shear rate region.It is considered that the collagen fibers generated from FBR distribute anisotropic and partially aligned in the shearing direction,other than the randomly distributed nematic phases in normal skin tissue.Both storage modulus(G')and loss modulus(G")of PVCF are dramatically lower than that of the normal skin tissue,showing its high flexibility and low mechanical properties.For the PVCF sample,there was a crossover between the storage modulus(G')and the loss modulus(G")in the high-frequency region,where G" was higher than G' with liquid-like behavior,reflecting the long-range fiber motions and the large-scale conformation rearrangement at high shear rates and indicating injectability of PVCF matrix,which is attractive for many in vivo applications such as minimally invasive surgery.During the tensile test,tensile strength of the PVCF was much lower than that of the skin sample,which is consistent with the rheological results,suggesting its high flexibility.In the cyclic tensile test,the energy dissipation of PVCF at the first circle was 11.9 KJ/m3,which is 2.9-fold of that in skin sample,suggesting higher viscoelastic property of PVCF.After 10 cycles,the recovery loss of tensile strength is 40.2%for PVCF and 25.2%for normal skin,suggesting less robustness of PVCF.The stress-relaxation test suggests significantly greater and faster decrease of stress under a constant strain,which indicates the PVCF matrix will endure less and shorter pressure during transplantation and contribute to less necrosis as a result.We can conclude that the highly flexible,super viscoelastic PVCF matrix is a mechanically suitable transplant site with less robustness and mechanical stress.Transplantation of neonatal mouse skin cells into PVCF matrix to improve the efficiency of hair regenerationAfter the removal of the silicone catheter at different time points,neonatal MECs and MDCs were delivered into the PVCF matrix for hair follicle regeneration in vivo.Dense black hair shafts were observed within cysts after transplantation.The number of hair follicles regenerated among different time point groups was highest at 3 weeks of FBR,as well as greater than that in traditional patch assay.Histological analysis revealed fully developed follicles of a normal size and structure.The number of necrotic sites was lower in transplanted mice than in control mice.Furthermore,the density of the newly formed,follicle-associated vascular structures was higher at 3 weeks than at the other time points.Conclusion(1)FBR-induced PVCF matrix composed of cross-linked collagen fibers that formed a porous mesh structure with newly generated vessels interconnected and inflammatory cells scattered inside.By manipulating the FBR duration,a preprogrammed PVCF matrix physiologically mimicking the graft site microenvironment can be achieved.(2)The mechanical properties of PVCF matrix are incredibly favorable for graft survival in comparison with that of skin sample.It revealed that the PVCF matrix as a transplant site mimics a super viscoelastic hydrogel that is highly flexible,with less robustness performance and rapid decreasing stress under the constant strain.(3)PVCF matrix generated in the middle stage of FBR process(2-3 weeks)would promote cell survival and inhibits apoptosis,and thus improve the HF regeneration.This suggested that our strategy of improving the graft site microenvironment could efficiently improve the success rate of transplantation.