Regenerative Medicine & Tissue Engineering Notes

Regenerative Medicine

Tissue Regeneration

  • Definition: Regrowth of damaged or missing organ parts from remaining tissue, leading to complete restoration.
  • Human Regeneration:
    • Adult humans can regenerate some organs (e.g., liver, skin).
    • Many tissues do not regenerate (e.g., cartilage, nerve tissue).

Tissue Engineering

  • Definition: Combines scaffolds, cells, and bioactive molecules to create functional tissues.
  • Evolved From: Biomaterials field.
  • Clinical Applications:
    • Structural replacement (e.g., cosmetic reconstruction).
    • Functional replacement (e.g., musculoskeletal tissues).
    • Wound healing (e.g., autologous skin grafts, chronic wounds, corneal replacement).

Regenerative Medicine vs. Tissue Engineering

  • Interchangeable Terms: Largely considered so.
  • Regenerative Medicine Scope: Broader, includes tissue engineering and research on self-healing using the body's systems, sometimes with foreign biological material.

Tissue Engineering: Key Ingredients

  • Cell precursors.
  • Bioactive molecules.
  • Cell Source:
    • Autologous: From the same individual.
    • Allogeneic (Heterologous): From a different individual of the same species.
    • Xenogeneic: From a different species.
  • Cell Precursors Role: Produce ECM and/or synthesize and release bioactive molecules (e.g., growth factors).

Origin and Types of Cell Precursors

  • Mesenchymal stem cells (MSCs).
  • Embryonic stem cells (ESCs).
  • Genetically manipulated cells (iPSC).
  • Native cells of a tissue (e.g., chondrocytes, skin fibroblasts).
  • Derived from healthy or pathological tissue.

Bioactive Molecules

  • Growth factors.
  • Enzymes.
  • Interleukins.
  • Anti-inflammatory molecules.
  • Tissue engineering signals.
  • Small molecules.
  • Mechanical forces.

Tissue Engineering: Scaffolds

  • Natural Products:
    • Purified collagens (usually Type I).
    • Fibronectin.
    • Fibrin.
    • Hyaluronan.
    • Decellularized extracellular matrix.
    • Silk.
    • Plant polysaccharides (e.g., cellulose).
    • Animal polysaccharides (e.g., Chitin).
  • Synthetic Biomaterials:
    • Polylactic acid (PLA) - biodegradable.
    • Polyglycolic acid (PGA) - biodegradable.
    • Carbon fibers.
    • Hydroxyapatite.
  • Composites (Synthetic/Natural):
    • PLGA/Collagen Type I.
    • PLA/Elastin.
    • PLGA/Hyaluronan.

The Tissue Engineering Triad

  • Cell Source:
    • Stem cells.
    • Primary cells and cell lines.
    • Organoids.
    • Genetic engineering tools.
    • DNA/RNA
  • Scaffolds:
    • Hydrogels.
    • Engineered Tissue Equivalents.
    • Tissue Arc.
    • Organs-on-chips.
    • 3D (cell) printing.
    • Nanoparticles
  • Niche Properties:
    • Growth factors, signalling molecules.
    • Proteins.

Application in Knee Joint Pathology

  • Knee Replacement Surgeries (USA): 790,000 annually.
  • Knee Replacement Surgeries (England & Wales, 2017): 106,000.
  • Tissue Engineering Offers: Treatment prior to joint replacement.

Common Knee Sports Injuries

  • Anterior Cruciate Ligament (ACL) injuries.
  • Collateral Ligament injuries.
  • Meniscal Cartilage Tears.
  • Cartilage lesions (e.g., Osteochondritis Dissecans).
  • All can lead to Osteoarthritis.

Osteochondrosis Dissecans (OCD)

  • Definition: Inflammation of bone and cartilage resulting in loose fragments.
  • Historical Context:
    • Alexander Monro (1738): Initially described.
    • Paget (1870): Described as a form of quiet necrosis.
    • Konig (1881): First to use the term osteochondritis dissecans.
  • Etiology: Multi-factorial: genetic predisposition, inflammation, spontaneous avascular necrosis, repetitive microtrauma.
  • Epidemiology:
    • Occurs in 15-29 per 100,000 patients.
    • More common in males (ratio 2:1 to 3:1).
    • Higher incidence in young athletes.
    • Common in 10-20 years age group.
    • Knee is most commonly affected joint.

Pathophysiology of OCD

  • Idiopathic focal joint disorder affecting subchondral bone.
  • Cartilage and subchondral bone fragment separates from articular surface.
  • Due to lack of blood supply (osteonecrosis) in underlying bone.
  • Decalcification of trabecular bone matrix.
  • Untreated OCD can lead to degenerative arthritis.

OCD Symptoms

  • Pain.
  • Inflammation, edema, swelling, and soreness.
  • Catching and locking in the joint.
  • Reduced range of motion.
  • Crepitus (grating, cracking, or popping sound).

OCD Diagnosis

  • Radiographic examination.
  • MRI.

Pioneering Research: Autologous Chondrocyte Transplantation (ACT)

  • Brittberg et al. (1994): Treatment of deep cartilage defects in the knee.
  • Publication: New England Journal of Medicine 331:889-895.

Autologous Chondrocyte Implantation (ACI) - First Generation

  • Biopsy of healthy cartilage.
  • Enzymatic digestion to release cells.
  • Cultivation for 11-21 days (10-fold increase in cell number).
  • Periosteal flap taken from medial tibia.
  • Trypsin treatment.
  • Injection of cultured chondrocytes under flap into lesion (2.6 x 10610^6 - 5 x 10610^6 cells).
  • Periosteal flap sutured over lesion.

Methodology Details (Brittberg et al., 1994)

  • 300-500 mg cartilage harvested from low weight-bearing region.
  • Cartilage digested with collagenase & deoxyribonuclease to release 180,000 to 455,000 cells.
  • Cells seeded at 5000-10,000 cells per cm2cm^2 in T25 or T75 flasks.
  • Maintained in media containing patient serum (autologous serum).
  • Full-thickness biopsy (to subchondral bone); 200-300mg harvested (mean 280mg, range 4-1700mg).
  • Mean number of cells/mg (500 Pts) - 2,600/mg.
  • 10 x 10ml Autologous Venous Blood taken
  • In vitro cell expansion in DMEM + 10% autologous serum
  • Initial culture: 25x25cm culture bottles - 1 week
  • Next cultured: 75x75cm culture bottles - 2 weeks
  • Then trypsinised and suspended at 30 million cells/ml
  • At ACT/ACI: implanted at 2 million cells/ square cm of defect
  • Periosteum provides source of growth factors & stem cells.
  • Autologous chondrocytes repair & remodel the cartilage ECM
  • Sutures & Fibrinogen/fibrin glue used to seal periosteum
  • Transplantation 12-14 days after initial surgery.
  • Chondrocytes trypsin treated, washed and resuspended in 50-100 µl volume 2.6- 5 million cells
  • Chondral lesion excised to surrounding normal tissue.
  • Cartilage defect covered with periosteal flap. Sutured to normal cartilage.
  • Periosteal flap potential source of growth factors and stem cells
  • Autologous Chondrocytes injected beneath the periosteal flap to initiate repair and remodeling of cartilage extracellular matrix

Results (Brittberg et al., 1994)

  • 26 patients (mean age 27 years) treated.
  • Defect size ranged from 1.6-6.6 cm.
  • Types of defects repaired:
    • Traumatic femoral cartilage defect: 16 patients
    • Osteochondral Lesions: 3 patients
    • Patellar cartilage defect: 7 patients
  • Femoral Condylar Defects in 16 Patients Treated with Transplanted Chondrocytes.
  • Two Years Post surgery 87.5% with femoral defects showed a good outcome.
  • 12-46 months postoperative
  • Biopsies, 15 biopsies in total
  • 73% hyaline-like cartilage
  • 27% fibro-cartilage

Theoretical Explanations for Repair Process

  1. Transplanted chondrocytes repopulate the defect area, producing new cartilage, and the periosteum seals the defect.
  2. Periosteum stimulates replication of transplanted chondrocytes, which produce new cartilage matrix.
  3. Periosteum and transplanted chondrocytes stimulate surrounding chondrocytes or cells in deep noncalcified/calcified zones to enter the defect, divide, and repair it.

Recap: First Generation ACT

  • Cell Precursors: Autologous chondrocytes from healthy cartilage (culture expanded ex vivo).
  • Natural Product: Periosteum - source of stem cells and growth factors, covering for defect.
  • Sutures: Sealing periosteal flap.
  • Modification: Use of fibrinogen and thrombin (autologous) to seal the periosteal piece over the defect.

Second Generation ACT

  • Collagen Type I/III scaffold.
  • Bilayer collagen membrane replaces periosteal flap.
  • Advantages:
    • Simplified surgical procedure (reduced surgical morbidity).
    • Reduced chondrocyte Hypertrophy.

Third Generation ACT

  • Biomaterials seeded with chondrocytes.
  • Trimmed to match defect size.
  • Implanted without periosteal flap.
  • Variations:
    • Use of mesenchymal stem cells.
    • MACI (Matrix-Induced Autologous Chondrocyte Implantation).

Alternative scaffolds for MACI

  • Hyaluronic-acid based scaffolds- Hyalograft
  • Polymers of polylactin and polyglactin- BioSeed-C and Novocart3D
  • Three dimensional collagen gel (Type I collagen) CaREs

Third/Fourth Generation – Animal models and Clinical Trails

  • One-stage focal cartilage defect treatment with bone marrow mononuclear cells and chondrocytes leads to better macroscopic cartilage regeneration compared to microfracture in goats
  • Cell precursors: allogenic stem cells, autologous stem cells
  • Scaffolds: hydrogel, fibrous scaffold, decelluraised ECM, or composite

Macroscopic Evaluation

  • The Mastbergen score
    • a four-point scale ranging from a macroscopically healthy and smooth cartilage surface (0 points) to cartilage degeneration characterized by deep grooves and surrounding cartilage damage (four points).
  • The International Cartilage Repair Society (ICRS) macroscopic evaluation of cartilage repair score.
    • This score (0–12 points scale) evaluates the macroscopic cartilage repair on degree of defect repair and fill, integration into border zone and macroscopic appearance. The higher the score the better the macroscopic cartilage repair.

Microscopic Evaluation

  • The O'Driscoll score.
    • This score (range 0–24 points) evaluates the regenerated cartilage on the amount of Safranin-O staining in the matrix, cellular morphology and clustering, the structural characteristics of the tissue and degenerative changes in adjacent tissue. The higher the score the better the microscopic cartilage regeneration.
  • The Mankin score
    • which ranges from normal appearing articular cartilage (0 points) to tissue with complete disorganization, no matrix staining and hypocellularity or cloning (14 points). Similar score processing from the two observers was performed as with the macroscopic scoring.

Conclusion

  • Treatment of focal articular cartilage lesions in goats using a combination of Mononuclear Fibroblast (MNF) cells from bone marrow and unexpanded chondrocytes leads to better macroscopic regeneration compared to microfracture.
  • However, the technique requires further fine-tuning to decrease the negative influence on other joint compartments.