Co-culture of primary chondrocytes and stem cells for regeneration of cartilage in the head and neck

Reconstruction of cartilaginous structures of the head and neck is a field that’s been traditionally limited by difficulties sourcing sufficient donor cartilage, post-operative infection and achieving consistent aesthetic and functional results. Bioprinting is a technique that utilises 3D printed scaffolding with embedded bioinks typically containing living cells, supporting structures and hydrogel matrices to create customized implants that could address some of the limitations of contemporary reconstructive techniques. The development and clinical translation of bioprinting into real word reconstructive surgery is an extensive ongoing task requiring the collaboration of scientists, engineers, and surgeons. In this thesis, we hope to better inform the choice of living cell types for cell-laden bioinks through answering two key questions: 1. Is there a role for co-culture of chondrocytes with stem cells as a method of expanding effective chondrocyte populations – helping to address limitations where sufficient donor cartilage is unavailable. 2. Are there any key differences between the harvest, digestion and proliferation of auricular and nasal septal chondrocytes – helping to better understand the impact of different donor sites on chondrocytes and its impact on clinical practice. Our thesis concludes that co-culture of chondrocytes with stem cells at the same cell density is at least equivalent to mono-culture of chondrocytes, supporting its use as a technique for expanding effective chondrocyte populations and reducing the quantity of donor cartilage required. This thesis also found both auricular and nasal septal cartilage could be safely harvested from the concha and nasal septum respectively and kept free of contamination prior to digestion utilising an antibiotic solution, with no significant differences in proliferation rates between the two types

The Role of a Novel Biosynthetic Graft in Nasal Reconstruction

Background: Septorhinoplasty is a common procedure, and when native septal cartilage is insufficient for grafting during reconstruction, a variety of alternatives are currently used with varying success. Thus, the aims of this thesis are threefold: 1) summarise the evidence for current alternative grafts, 2) identify emerging alternatives, and 3) assess the feasibility of a novel hybrid graft in nasal reconstruction. Methods: The first chapter of this thesis establishes a foundational understanding of the anatomy and function of the nose and septorhinoplasty. This contextualises the second chapter which utilises a systematic review of the literature to qualitatively evaluate complication rates of current alternative grafts. Furthermore, emerging alternatives are summarised. One of these emerging grafts, a hybrid bioactive-polymer scaffold (actv-graft®), is then used in a feasibility study in three sheep after ethics approval and grant acquisition in chapter 3. Results: The results of six systematic reviews were qualitatively synthetised in chapter 2. Biologic grafts were generally associated with higher rates of warping, revision surgery, donor-site morbidity, and resorption. Synthetic grafts were more commonly associated with extrusion, infection, and removal surgery. No single alternative graft was ideal. Tissue-engineering and hybrid bioactive-polymer scaffolds were identified as two emerging fields. In chapter 3, actv-graft® was found to be biocompatible, biodegradable, have favourable volume augmentation over time, and stimulate host infiltration within the graft. Conclusion: Cost-effective, easily procured, patient-specific, bioactive, and biodegradable scaffolds are required in septorhinoplasty. This thesis establishes the feasibility of a promising graft. However, a longer and larger cohort animal study would aid in characterising the safety profile, chondrogenic potential, and degradation rate of actv-graft® prior to human trials

Randomised controlled clinical trial comparing the clinical outcome of autologus fascia lata to synthetic mesh for abdominal sacrocolpopexy among successfully repaired vault prolapse patients

INTRODUCTION: Vaginal vault prolapse has been defined by the International Continence Society as descent of the vaginal cuff below a point that is 2 cm less than the total vaginal length above the plane of the hymen. Coexisting pelvic floor defects like cystocoele, rectocele or enterocele may be present in 72% of patients with vault prolapse. Pelvic organ prolapse has negative impact on quality of life of these women due to associated urinary, faecal and coital dysfunction. It is important to assess the defects of the different vaginal compartment and counsel these women before planning surgical management. A clear understanding of the supporting mechanisms of the uterus and the vagina are important in order to make the right choice of the corrective procedure and also to minimise the risk of occurrence of vault prolapse. SCOPE OF THE STUDY: Management of vaginal vault prolapse may be complex and surgical reconstruction can be challenging. The most appropriate operative procedure and approach should be selected to achieve an optimal result for the patient with vaginal vault prolapse. In the literature, several vaginal and abdominal procedures have been described to treat vault prolapsed and there is no consensus on the most effective approach or technique. Evidence shows that abdominal repair yields better long terms results with reduced incidence of recurrence. Sacrocolpopexy is a valid technique to treat apical and anterior vaginal wall prolapse. Abdominal sacrocolpopexy using synthetic mesh has proven its worth overtime. METHODOLOGY: Study Design: It is a prospective, single blinded, randomised controlled clinical trial. Sample Setting: It is a single blinded randomised controlled clinical trial (RCT) performed to compare the clinical outcome of autologous fascia lata over synthetic mesh in successfully repaired cases of vault prolapse by abdominal sacrocolpopexy. The study was conducted in the department of Urogynaecology, Institute of Social Obstetrics and Government Kasturba Gandhi Hospital for Women and Children, Madras Medical College, a tertiary care Hospital in Chennai, India. The study group comprised predominantly patients belonging to the low socio-economic status. The study population was mostly the referral patients with clinical evidence of vaginal vault prolapse. Duration of the Study: The study was conducted from April 2009 to March 2012. Sample: 58 Vaginal vault prolapse patients (N=58) were included in this study. Out of this 28underwent abdominal sacrocolpopexy with autologous Fascia Lata and 30 with Synthetic Mesh. Inclusion Criteria: Women with stage 3 to stage 4 vaginal vault prolapse (primary) or recurrent vaginal vault prolapse scheduled for abdominal sacrocolpopexy were included. Exclusion criteria: 1. Stage 1 to 2 vault prolapse. 2. Vaginal vault prolapse with abnormal pap smear. 3. Comorbid medical diseases like valvular heart disease, ischaemic heart disease, chronic obstructive pulmonary disease, renal / liver disease and central nervous system disorders. 4. BMI more than 30. SUMMARY AND CONCLUSIONS: Blood loss, duration of surgery & hospital stay did not differ significantly in the two groups. ANATOMICAL SUCCESS: Amongst the successfully repaired patients using autologus fascia lata, objective anatomical success rates were superior with reference to POP Q points Aa, Ba, Ap, Bp compared to synthetic mesh. However the anatomical outcome of C point in fascia lata group was comparable to that of mesh group. FUNCTIONAL SUCCESS: In the current study post operative resolution of lower urinary tract symptoms was equal in both fascia lata and synthetic mesh group. URINARY SYMPTOMS: None in the fascialata group developed UTI however3.6% in synthetic mesh group developed urinary tract infection in the postoperative follow up. Combining Burch colposuspension along with ASCP for patients who had coexisting SUI (overt and occult) and vault prolapse has relieved SUI in both groups. The number of participants who developed denovo SUI was 10.7% in fascia lata group and 3.3% in mesh group. None developed denovo urgency and urge urinary incontinence were same in both groups. BOWEL SYMPTOMS: Complete resolution of all pre operative bowel symptoms occurred in the autologous fascia lata group incontrast to the mesh group where 3% reported faecal urgency in the postoperative follow up. SEXUAL SYMPTOM: With fascia lata suspension, none developed de novo dyspareunia unlike mesh suspension. Nevertheless in the synthetic mesh group none developed mesh exposure / extrusion or infection post operatively. In the fasci lata group, there was no significant postoperative morbidity like haematoma infection and muscle herniation from the harvested site. The current study has revealed that vault prolapse repair by abdominal sacrocolpopexy using autologous fascia lata has comparable anatomical success to synthetic mesh. The functional outcome (clinical success) was better with autologous fascia lata suspension compared to mesh suspension. • This study highlights that autologous fascia lata is a versatile graft with comparable efficacy to mesh. Offers good and durable mechanical support of the vaginal vault. In future, larger and prospective, randomized clinical trials and a long-term follow-up are needed to further evaluate durability, anatomical outcomes and patient satisfaction after ASC with autologous fascia lata. RECOMMENDATIONS: Autologous Fascia lata is strong, pliable and homologous tissue does not involve foreign body reaction or infection. After repetitive notifications of FDA about nonuse of mesh in pelvic floor surgeries, there is increasing scope of biological grafts in reconstructive surgeries. According to this study, autologous fascia lata compares favorably in efficacy to monofilament, polyprophylene mesh and it is not associated with any significant morbidity. Follow up in our series of patients is adequate to assess accurately treatment efficacy, harvest site morbidity and patient satisfaction. There had been good medium term followup results in our study and patient satisfaction with the procedure is high. The successfully operated patients who were completely asymptomatic (in both groups) reported high patient satisfaction rate. The following advantages of autologus fascia lata has been proved in this study: • It is a scientific proven operation which is independent of synthetic graft and is devoid of mesh related problems and other surgery related complications like infection, extrusion, chronic vaginal pain, constipation and dyspareunea. • Because of minimal complications , abdominal sacro colpopexy using fascia lata can be offered to patients who hail from remote villages with no access for followup. • Autologous graft is readily available with the patient and the patient is not dependant on any commercial material for suspension. The anatomical and functional outcomes of autologus fascia lata are equivalent to that of synthetic mesh in abdominal sacrocolpopexy for management of vault prolapse, Hence abdominal sacral colpopexy using autologous fascia lata may be considered primarily for women with vaginal vault prolapsed

sj-jpg-1-aor-10.1177_00034894211058115 – Supplemental material for Cost Utility Analysis of Costal Cartilage Autografts and Human Cadaveric Allografts in Rhinoplasty

Supplemental material, sj-jpg-1-aor-10.1177_00034894211058115 for Cost Utility Analysis of Costal Cartilage Autografts and Human Cadaveric Allografts in Rhinoplasty by Nicole C. Starr, Liza Creel, Christopher Harryman and Nikita Gupta in Annals of Otology, Rhinology & Laryngology</p

PRELIMINARY FINDINGS OF A POTENZIATED PIEZOSURGERGICAL DEVICE AT THE RABBIT SKULL

The number of available ultrasonic osteotomes has remarkably increased. In vitro and in vivo studies have revealed differences between conventional osteotomes, such as rotating or sawing devices, and ultrasound-supported osteotomes (Piezosurgery®) regarding the micromorphology and roughness values of osteotomized bone surfaces. Objective: the present study compares the micro-morphologies and roughness values of osteotomized bone surfaces after the application of rotating and sawing devices, Piezosurgery Medical® and Piezosurgery Medical New Generation Powerful Handpiece. Methods: Fresh, standard-sized bony samples were taken from a rabbit skull using the following osteotomes: rotating and sawing devices, Piezosurgery Medical® and a Piezosurgery Medical New Generation Powerful Handpiece. The required duration of time for each osteotomy was recorded. Micromorphologies and roughness values to characterize the bone surfaces following the different osteotomy methods were described. The prepared surfaces were examined via light microscopy, environmental surface electron microscopy (ESEM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM) and atomic force microscopy. The selective cutting of mineralized tissues while preserving adjacent soft tissue (dura mater and nervous tissue) was studied. Bone necrosis of the osteotomy sites and the vitality of the osteocytes near the sectional plane were investigated, as well as the proportion of apoptosis or cell degeneration. Results and Conclusions: The potential positive effects on bone healing and reossification associated with different devices were evaluated and the comparative analysis among the different devices used was performed, in order to determine the best osteotomes to be employed during cranio-facial surgery

3D Bioprinting of Hyaline Cartilage using Nasal Chondrocytes

Hyaline cartilage is a strong and flexible connective tissue found throughout the human body. It provides important structural and functional support for the nose, ribs, larynx, and trachea. Due to the avascular nature of the hyaline cartilage, its defects or lesions are non-regeneratable and non-healing, which can progress into diseases such as osteoarthritis (OA) or nasal airway obstruction and result in severe clinical complications. Cell-based cartilage tissue engineering using 3D bioprinting techniques can generate functionalized cartilage replacement in vitro using autologous cells, which can become a promising prospective treatment for these defects and lesions. The 3D bioprinting techniques allow on-demand fabrication of engineered and patient-specific cartilage tissue to replace damaged tissue and restore normal cartilage functions. However, existing 3D bioprinting research primarily focuses on the formulation and engineering of biomaterials, yet lacks thorough biochemical evaluations and substantial evidence to indicate the clinical potential of the bioprinted cartilage. To address this challenge, this thesis focuses on 3D bioprinting and regeneration of hyaline cartilage using naturally derived polymer, with in-depth evaluations of appropriate cell sources, in vitro, and in vivo biochemical and biomechanical performance of the engineered tissue. Chapter 1 introduces the research topic with a review of hyaline cartilage structure, biochemical and biomechanical properties, potential cell source for engineered cartilage tissue, and suitable bioink materials used for 3D bioprinting. This review suggests that nasal chondrocytes are among the most suitable cell sources for hyaline cartilage regeneration. Chapter 2 provides insights into the rheological and viscoelastic properties of collagen and their roles in micro-extrusion bioprinting. The first project (Chapter 3) describes the 3D bioprinting of cartilage tissue using nasal chondrocytes laden type I collagen as bioink. A biomimicry shape with non-cytotoxicity was generated using a state-of-the-art freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting technique. The engineered cartilage showed comparable biochemical properties to native nasal cartilage. The mechanical characterization and in vivo stability of the engineered nasal cartilage substitutes were needed to further support its potential for clinical application and formation of patient-specific surgical-ready shapes. Therefore, the second project (Chapter 4) extends this study and investigates chondrogenic culture's effects on the biochemical and mechanical properties of bioprinted constructs of nasal chondrocytes in vitro and in vivo in nude mice. Engineered nasal cartilage from nasal chondrocytes seeded on clinically approved type I/III collagen membrane scaffolds (Chondro-Gide) served as a control. The results showed excellent in vitro and in vivo performances comparable to those of clinically approved scaffolds. To further improve the printability and the shape integrity of collagen bioink, the third project (Chapter 5) used pre-crosslinked methacrylate collagen with thiolate hyaluronic acid and Poly(ethylene glycol) diacrylate (PEGDA) crosslinker as a bioink to investigate the printability and in vitro chondrogenesis. This novel bioink showed significantly improved printability, shape and size retention, and biochemical indicators compared to a collagen-only bioink. This thesis extensively studied 3D bioprinting and regeneration of hyaline cartilage using type I collagen-based hydrogels with nasal chondrocytes. The study results pave the way for the clinical application of 3D bioprinting to treat cartilage defects

Cost Utility Analysis of Costal Cartilage Autografts and Human Cadaveric Allografts in Rhinoplasty

Background: Human cadaveric allograft (HCA) and costal cartilage autograft (CCA) have been described for reconstruction during rhinoplasty. Neither are ideal due to infection, resorption, and donor site morbidity. The clear superiority of 1 graft over the other has not yet been demonstrated. This study assesses comparative costs associated with current grafting materials to better explore the cost ceiling for a theoretical tissue engineered implant. Materials and methods: A cost utility analysis was performed. Initial procedure costs include physician fees (CPT 30420), hospital outpatient prospective payments, ambulatory surgical center payments, and fees for the following: rib graft (CPT 20910), hospital observation, and DRG (155) for inpatient admission. Additional costs for revision procedure, included the following fees: physician (CPT 30345), rib graft, hospital outpatient prospective payment, and ambulatory surgical center payments. Total costs under each scenario were calculated with and without the revision procedure. Comparison of total costs for each potential outcome to the estimated health utility value allowed for comparison across rhinoplasty subgroups. Results: The mean cost of primary outpatient rhinoplasty using HCA and CCA were $8075 and$8342 respectively. Revision outpatient rhinoplasty averaged $7447 and increased to$8228 if costal cartilage harvest was required. Hospital admission increased the cost of primary rhinoplasty with CCA to $8609 for observational admission and to$13653 for 1 day inpatient admission. Revision CCA rhinoplasty with an inpatient admission complicated by pneumothorax increased costs to $21 099. Conclusion: Cost of rhinoplasty without hospitalization was similar between HCA and CCA and this cost represents the lower limit of a practical cost for an engineered graft. Considering complications such as need for revision or for admission after CCA due to surgical morbidity, the upper limit of cost for an engineered implant would approximately double. </jats:sec

Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture

The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels