Abstract:

Chondrocytes with remnants of nuclei and biomolecules were recently reported in two Cretaceous dinosaurs from North America and China. For multiple reasons, it was hypothesized that calcified cartilage (CC) had a better potential than bone to preserve ancient cells. Here we provide the first experimental test to this hypothesis by focusing on the most important variable responsible for cellular preservation: the postmortem blockage of autolysis. We compare the timing of autolysis between chondrocytes and osteocytes in an avian model (Anas platyrhynchos domesticus) buried for up to 60 days under natural conditions that did not inhibit autolytic enzymes. Within 15 days post-burial, almost all osteocytes were already cytolyzed but chondrocytes in CC were virtually unaffected. All osteocytes were cytolyzed after 30 days, but some chondrocytes were still present 60 days post-burial. Therefore, even in harsh conditions some CC chondrocytes still survive for months postmortem on a time scale compatible with permineralization. This is consistent with other data from the forensic literature showing the extreme resistance of hyaline cartilage (HC) chondrocytes after death and does support the hypothesis that CC has a better potential than bone for cellular preservation, especially in fossils that were not permineralized rapidly. However, because the samples used were previously frozen, it is possible that the pattern of autolysis observed here is also a product of cell death due to ice crystal formation and not strictly autolysis, meaning a follow-up experiment on fresh (non-frozen samples) is necessary to be extremely accurate in our conclusions. Nevertheless, this study does show that CC chondrocytes are very resistant to freezing, suggesting that chondrocytes are likely better preserved than osteocytes in permafrost fossils and mummies that underwent a freezing-thawing cycle. It also suggests that cartilage (both hyaline and calcified) may be a better substrate for ancient DNA than bone. Moreover, even though we warrant follow-up taphonomy experiments with non-frozen samples paired with DNA sequencing, we already urge ancient DNA experts to test CC as a new substrate for ancient DNA analyses in fossils preserved in hot and temperate environments as well.

Key words: experimental taphonomy, chondrocytes, osteocytes, postmortem autolysis, freeze related cell-death, cellular and biomolecule fossilization, aDNA substrate

摘要：

近期，在发现于美国和中国的两例早白垩世恐龙中报道了保存有细胞核物质和生物分子的软骨细胞的存在。基于多种原因，研究人员认为，钙化软骨比骨骼更有可能保存远古细胞。通过针对影响细胞保存的最主要因素：机体死亡后细胞自溶过程的停止，对这一假说进行了首次实验验证。以家鸭 (Anas platyrhynchos domesticus)为模型，比较了在不抑制自溶酶的情况下，自然埋藏60天内，软骨细胞和骨细胞的自溶过程。埋藏后的15天内，几乎所有骨细胞均发生了自溶，而钙化软骨细胞则基本未受影响。埋藏30天后，所有骨细胞均已自溶，但一些软骨细胞在埋藏60天后仍然存在。因此，即使在恶劣的条件下，钙化软骨细胞仍然能够在动物机体死亡后存活数月之久，而这一时间足以实现矿化过程。这一结果与一些法医文献中的数据相吻合，表明透明软骨细胞在机体死亡后能够长时间抵抗分解，且支持了钙化软骨比骨骼更有保存细胞结构的潜力的假说，尤其是在未能快速矿化的化石中。然而，由于所使用的标本预先经过冷冻，所观察到的自溶模式也有可能是细胞由于冰晶形成而死亡的结果，而非严格的自溶过程，因此有必要进一步对新鲜标本开展实验以提高结论的准确度。无论如何，研究明确显示透明软骨和钙化软骨的软骨细胞受到冷冻的影响比骨细胞更小。这暗示软骨细胞比骨细胞更可能在发现于永久冻土的化石或木乃伊中保存下来，而软骨(包括透明软骨和钙化软骨)可能是比骨骼更为理想的古DNA研究对象。尽管有必要对未经冷冻的新鲜样品进行后续实验并辅以DNA测序，建议古DNA专家将保存在热带和温带环境的化石中的钙化软骨作为古DNA分析的新对象。

关键词: 实验埋藏学, 软骨细胞, 骨细胞, 死后自溶, 冷冻相关细胞死亡, 细胞和生物分子石化, 古DNA研究对象