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1. Haemostasis (minutes): vasoconstriction, platelet plug and fibrin clot. Platelets release growth factors (PDGF, TGF-beta, VEGF). 2. Inflammation (days 1-4): neutrophils clear bacteria, then macrophages remove debris and release cytokines and growth factors. 3. Proliferation (days 4-21): fibroblasts make collagen and granulation tissue, angiogenesis restores blood supply, keratinocytes migrate to close the epithelium (re-epithelialisation), and myofibroblasts contract the wound. 4. Remodelling (weeks to years): type III collagen is replaced by type I, cross-linked and reorganised; the scar reaches about 80 percent of the original tissue strength. The extracellular matrix (ECM) provides structural support, binds growth factors and gives signals to cells; its composition guides cell behaviour.

Regeneration replaces lost cells with the same cell type and restores structure and function (liver, epidermis, bone, the lining of the gut). Repair (fibrosis) fills the defect with connective tissue and leaves a scar with reduced function (myocardium, central nervous system, cartilage). Factors: proliferative capacity of the cells (labile, stable and permanent tissues), the extent of injury (an intact basement membrane favours regeneration), inflammation and infection, blood supply, and mechanical environment.

1. List the phases of wound healing. 2. Which cells make granulation tissue? 3. What is the difference between repair and regeneration? 4. Name three growth factors released by platelets.

1. Haemostasis, inflammation, proliferation, remodelling. 2. Fibroblasts (with endothelial cells forming new vessels). 3. Regeneration restores the original tissue; repair replaces it with a scar. 4. PDGF, TGF-beta and VEGF.

Potency: totipotent (zygote; can form the whole organism including placenta), pluripotent (embryonic stem cells and induced pluripotent stem cells; all three germ layers), multipotent (adult stem cells; a limited family of cell types), unipotent. Sources: embryonic stem cells (ethical issues and tumour risk), adult stem cells (bone marrow, fat, skin, gut) and induced pluripotent stem cells (reprogrammed from somatic cells with Oct4, Sox2, Klf4 and c-Myc; patient-specific, avoiding rejection).

Hematopoietic stem cells (HSC) in the bone marrow give all blood cells and are used in bone marrow transplantation. Mesenchymal stem cells (MSC, from bone marrow, adipose tissue and umbilical cord) differentiate into bone, cartilage and fat, and secrete anti-inflammatory and trophic factors. They have low immunogenicity.

The stem cell niche is the microenvironment (neighbouring cells, ECM, growth factors and mechanical cues) that maintains stem cells. Homing is the migration of transplanted cells to injured tissue guided by chemokines (for example SDF-1 with CXCR4). Secretome: the mixture of growth factors, cytokines and extracellular vesicles (exosomes) released by cells. Much of the benefit of MSC therapy is paracrine, which has led to cell-free approaches using vesicles.

1. Define pluripotent and multipotent. 2. How are induced pluripotent stem cells made? 3. What are two functions of MSCs? 4. What is the stem cell niche?

1. Pluripotent: can form all three germ layers; multipotent: can form a limited range of related cell types. 2. By reprogramming somatic cells with factors such as Oct4, Sox2, Klf4 and c-Myc. 3. Differentiation into bone, cartilage and fat, and secretion of anti-inflammatory and trophic factors. 4. The microenvironment that maintains stem cells.

A biomaterial is a material designed to interact with biological systems. History progressed from inert materials to bioactive and biodegradable ones. - Synthetic polymers: PLA, PGA and PLGA (biodegradable polyesters), PCL, polyethylene glycol. Controllable properties, but lack cell recognition sites. - Natural materials: collagen, gelatin, fibrin, hyaluronic acid, alginate, chitosan, silk, decellularised ECM (dECM). Good bioactivity, variable batches and weaker mechanics. - Ceramics: hydroxyapatite and tricalcium phosphate for bone. - Hydrogels: water-swollen networks resembling soft tissue. Design requirements: porosity and interconnected pores for cells and vessels, degradation matched to tissue formation, appropriate stiffness and topography (cells respond to substrate stiffness, mechanotransduction) and non-toxic degradation products.

Techniques: electrospinning (nanofibres resembling ECM), solvent casting and particulate leaching, freeze drying, and 3D printing (CAD/CAM design from imaging data allows patient-specific scaffolds; bioprinting deposits cells together with hydrogel bioinks).

Biocompatibility is the ability of a material to perform its function without causing an adverse host response. The ISO 10993 series defines biological evaluation of medical devices (cytotoxicity, sensitisation, irritation, systemic toxicity, genotoxicity, implantation, haemocompatibility). In vitro tests: cytotoxicity with extracts or direct contact, and viability assays (MTT, Live/Dead staining, LDH release). Animal models follow the 3Rs principle: replacement, reduction and refinement. Reconstructed tissues and organoids reduce animal use.

1. Compare synthetic and natural biomaterials. 2. Which scaffold property influences cell fate through mechanotransduction? 3. What does ISO 10993 cover? 4. What are the 3Rs?

1. Synthetic: tunable and reproducible but lack recognition sites. Natural: bioactive but variable and mechanically weaker. 2. Substrate stiffness (and topography). 3. Biological evaluation of medical devices (cytotoxicity, sensitisation, toxicity, implantation and others). 4. Replacement, reduction and refinement of animal use.

Growth factors: TGF-beta (matrix production, chondrogenesis), BMPs (bone), VEGF (angiogenesis), FGF, PDGF, EGF (epithelium). Delivery from scaffolds must control dose and timing. Platelet-rich plasma (PRP) is an autologous source of growth factors. Mechanical stimuli: compression for cartilage, stretch for tendon and muscle, fluid shear for vessels and bone. Bioreactors are devices that provide controlled culture conditions (nutrient perfusion, oxygen, mechanical loading) so that thicker constructs can be grown; types include spinner flask, rotating wall and perfusion bioreactors.

Skin: cultured epidermal autografts, dermal substitutes (collagen-glycosaminoglycan matrices, dECM) and bilayer constructs; bioprinting of skin with keratinocytes and fibroblasts. Chronic wounds (diabetic ulcers) benefit from PRP and smart dressings that sense pH, temperature or infection and release drugs. The scratch assay tests cell migration in vitro. Periodontium and oral mucosa: regeneration of the ligament, cementum and alveolar bone, and mucosal grafts made from cultured oral keratinocytes.

1. Name growth factors for bone and for angiogenesis. 2. What is a bioreactor used for? 3. What is PRP? 4. Which cells make up bioprinted skin?

1. BMPs for bone; VEGF for angiogenesis. 2. To grow larger constructs under controlled perfusion, oxygen and mechanical stimulation. 3. Platelet-rich plasma, an autologous concentrate of platelets and growth factors. 4. Keratinocytes and fibroblasts in a matrix.

Ossification: intramembranous (flat bones) and endochondral (long bones, through a cartilage template). Fracture healing phases: haematoma and inflammation, soft callus (cartilage), hard callus (woven bone) and remodelling into lamellar bone. Diamond concept for bone repair: osteogenic cells, osteoinductive signals (BMPs), an osteoconductive scaffold and mechanical stability, all supported by vascularity. Osteoconduction is growth of bone onto a scaffold surface; osteoinduction is recruitment and differentiation of progenitors; osseointegration is direct bone-implant contact.

Hyaline cartilage: chondrocytes in a matrix of type II collagen and proteoglycans (aggrecan), no blood vessels, nerves or lymphatics. Injuries do not heal spontaneously and often lead to osteoarthritis (OA), a degenerative joint disease; rheumatoid arthritis (RA) is an autoimmune synovitis. Repair strategies: microfracture (produces fibrocartilage), autologous chondrocyte implantation, osteochondral grafts, and scaffold-based engineering. The growth plate is a cartilage zone responsible for longitudinal bone growth in children.

Skeletal muscle: satellite cells activate, proliferate and fuse to repair fibres; large defects (volumetric muscle loss) need scaffolds. Tendons and ligaments: dense collagen I with few cells and poor blood supply; healing produces disorganised scar with reduced strength. Specialised junctions: the myotendinous junction and the enthesis (tendon-to-bone, a graded transition that is hard to reproduce).

Heart: cardiomyocytes divide very little after birth, so infarcts heal by scar. Approaches: cell therapy, engineered heart patches, and reprogramming of fibroblasts into cardiomyocytes. Nerves: the peripheral nervous system regenerates after injury (Schwann cells form bands of Bungner and guide axons), and nerve conduits bridge gaps. The central nervous system does not regenerate well because of inhibitory molecules from myelin and the glial scar.

1. Describe the diamond concept. 2. Why does cartilage heal poorly? 3. What is the role of satellite cells? 4. Compare PNS and CNS regeneration. 5. What is the enthesis?

1. Osteogenic cells, osteoinductive signals, an osteoconductive scaffold and mechanical stability, supported by vascularity. 2. It is avascular with few cells and a dense matrix. 3. They repair skeletal muscle by activating, proliferating and fusing with fibres. 4. PNS regenerates through Schwann cells; CNS regeneration is inhibited by myelin-associated molecules and the glial scar. 5. The transition zone where tendon or ligament attaches to bone.

2D culture on plastic is simple, cheap and reproducible but cells flatten, lose their phenotype and lack tissue-like gradients. 3D cultures (in scaffolds, hydrogels or as spheroids) restore cell-cell and cell-matrix interactions and gradients of oxygen and nutrients. Organoids self-organise from stem cells into mini-organs. Organ-on-chip devices combine microfluidics and cells to reproduce fluid flow and mechanical forces (lung, gut, liver). Applications: drug testing, disease models (osteoarthritis osteochondral models, pancreas models) and reducing animal experiments.

1. List two advantages of 3D over 2D culture. 2. What is an organ-on-chip? 3. What are organoids?

1. More realistic cell-cell and cell-matrix interactions, and physiological gradients of oxygen and nutrients. 2. A microfluidic device with cells that reproduces the microenvironment and mechanical forces of an organ. 3. Self-organising three-dimensional structures grown from stem cells that mimic organs.