Apoptosis and Cell Death in Orthopaedics

Two ways for a cell to die. Apoptosis is programmed cell death: an active, energy-dependent and tightly regulated process, often called "cell suicide". Necrosis is passive, unregulated death from injury.

• Apoptosis - cell shrinkage, chromatin condensation, DNA fragmentation and membrane blebbing, ending in apoptotic bodies that are phagocytosed with no inflammation; usually single cells
• Necrosis - cell swelling and membrane rupture, with spilled contents that provoke inflammation; usually groups of cells

Caspases do the work. Apoptosis is executed by caspases (cysteine-aspartate proteases). Two upstream pathways converge on the executioner caspases (3, 6, 7), which dismantle the cell and produce the characteristic shrinkage, blebbing and apoptotic bodies.

The intrinsic (mitochondrial) pathway. Internal stress - DNA damage, hypoxia, oxidative or ER stress, growth-factor withdrawal - shifts the balance of the Bcl-2 family towards pro-apoptotic Bax/Bak and away from anti-apoptotic Bcl-2; p53 is a key trigger. Bax and Bak permeabilise the mitochondrial outer membrane to release cytochrome c, which assembles the apoptosome with Apaf-1 and activates caspase-9.

The extrinsic (death-receptor) pathway. Death ligands - Fas ligand, TNF and TRAIL - bind their cell-surface death receptors and form a death-inducing signalling complex that activates caspase-8. Caspase-8 and caspase-9 then both activate the executioner caspases.

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The "eat me" signal. The absence of inflammation has a mechanism, and the examiner wants it. The dying cell keeps its membrane intact and flips phosphatidylserine from the inner to the outer leaflet, where it acts as an "eat me" signal.

Efferocytosis. Macrophages and neighbouring cells recognise the externalised phosphatidylserine and rapidly engulf the apoptotic cell and its bodies (efferocytosis) before the membrane ruptures, so the pro-inflammatory intracellular contents are never released. The whole sequence of shrinking, blebbing and fragmenting into apoptotic bodies takes hours and ends before inflammation can start. Efferocytosis is itself anti-inflammatory: it releases TGF-beta and IL-10.

The contrast with necrosis. In necrosis the membrane ruptures and DAMPs (danger signals) spill out, provoking inflammation. This is why apoptotic chondrocyte or osteocyte death is "quiet", whereas necrosis incites an inflammatory and reparative response.

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Why the assays matter. Recognising the morphology is only half the basic-science question; you also need to know how apoptosis is demonstrated, because distinguishing it from necrosis in cartilage, bone or a research model depends on these assays. They let you quantify apoptosis, for example counting apoptotic chondrocytes in osteoarthritic cartilage or apoptotic osteocytes in osteonecrotic or glucocorticoid-treated bone. That is how the disease associations in this topic were established and how candidate therapies are tested.

Each morphological feature has a matching assay.

• DNA fragmentation - the TUNEL assay labels the cut DNA ends in situ, and DNA laddering on gel electrophoresis shows internucleosomal cleavage as regular fragments in multiples of about 180 base pairs
• Phosphatidylserine externalisation - annexin V binding on flow cytometry, paired with a viability dye to separate early apoptosis from necrosis. Annexin V positivity with an intact membrane is early apoptosis; add propidium iodide to catch the late, leaky cells
• Active executioner caspase - cleaved caspase-3 immunostaining
• Morphology - electron microscopy (condensed chromatin, apoptotic bodies) and light microscopy (pyknosis, karyorrhexis). Blebbing under the microscope is the oldest assay of all

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The growth plate. Apoptosis of hypertrophic chondrocytes in the hypertrophic zone is an essential step in endochondral ossification. The cells die and are replaced by invading bone, which allows longitudinal growth.

Limb development. Interdigital apoptosis removes the webbing between the digits to separate the fingers and toes. Failure of this programmed cell death contributes to syndactyly.

Bone remodelling. Apoptosis participates in normal osteoblast and osteocyte turnover, and osteocyte apoptosis, the point most worth carrying, directs remodelling. The osteocyte network senses strain; where microdamage severs osteocyte processes, the affected osteocytes undergo apoptosis and the apoptotic signal recruits osteoclastic resorption to that site. This is targeted remodelling, and it is why fatigue damage is repaired where it occurs rather than at random. Two corollaries follow:

• Anything that suppresses osteocyte apoptosis - bisphosphonates and oestrogen both do - preserves the osteocyte network but also blunts the targeting signal, which is part of the argument about accumulated microdamage during prolonged antiresorptive therapy
• Anything that provokes osteocyte apoptosis over a wide area at once - ischaemia, corticosteroids - calls for remodelling on a scale the blood supply may not support, which is the bridge from this cell biology to osteonecrosis

Osteoarthritis. Excessive chondrocyte apoptosis contributes to cartilage matrix loss and joint degeneration.

Osteonecrosis and bone loss. Osteocyte apoptosis from ischaemia, steroids or alcohol is central to the pathogenesis of osteonecrosis (avascular necrosis). Glucocorticoids and disuse also drive osteocyte and osteoblast apoptosis and bone loss.

Other tissues. Apoptosis is implicated in tendon degeneration, intervertebral disc degeneration and aspects of fracture healing.

A therapeutic lever. Chemotherapy and radiotherapy kill tumour cells largely by inducing apoptosis, and tumours resist by evading it, for example through p53 loss or Bcl-2 overexpression. Bisphosphonates act partly by promoting osteoclast apoptosis, which reduces resorption, while reducing osteocyte and osteoblast apoptosis.